Hematology

LECTURE/PRELIMS/MS. AGNES GUZMAN

S

PLATELET PRODUCTION, STRUCTURE, AND FUNCTION with differentiation to the red blood cell line in another
arm, called erythropoiesis.
TOPIC OUTLINE
1 Megakaryocytopoiesis
2 Platelets
3 Platelet Ultrastructure
4 Platelet Activation
5 Platelet Activation Pathways

MEGAKARYOCYTOPOIESIS
(1) Megakaryocytes
(2) Megakaryocyte Differentiation and Progenitors
(3) Endomitosis
(4) Terminal Megakaryocyte Differentiation
(5) Megakaryocyte Membrane Receptors and Markers
(6) Thrombocytopoiesis: Platelet Shedding
(7) Hormones and Cytokines of Megakaryocytopoiesis

MEGAKARYOCYTES
→Unique bone marrow precursor cells of platelets.
→These are the largest cells in the bone marrow and are
polyploid, possessing multiple chromosome copies.
→Have a unique series of maturation
→On a Wright-stained bone marrow aspirate smear, each
megakaryocyte is 30-50 mm in diameter with a
multilobulated nucleus and abundant granular cytoplasm.
3 MEGAKARYOCYTE LINEAGE-COMMITTED
→Account for >0.5% of all bone marrow cells, and on a
PROGENITOR STAGES FROM THE CMP
normal Wright-stained bone marrow aspirate smear the
microscopist may identify 2-4 megakaryocytes per 10×lpf Burst-Forming Unit (BFU-Meg)
1
→In healthy intact bone marrow tissue, megakaryocytes, →Least mature
under the influence of an array of stromal cell cytokines, Colony Forming Unit (CFU-Meg)
2
cluster with hematopoietic stem cells in vascular niches →Intermediate colony
adjacent to venous sinusoid endothelial cells Light-Density CFU (LD-CFU-Meg)
3
→Responding to the growth factor thrombopoietin (TPO), →More mature
megakaryocyte progenitors are recruited from common →These are defined by their in vitro culture colony
myeloid progenitors (CMP) and subsequently differentiate characteristics.
through several maturation stages. →All three progenitor stages resemble lymphocytes and
→They extend proplatelet processes, projections that cannot be distinguished by Wright-stained light
resemble strings of beads, through or between the microscopy.
endothelial cells and into the venous sinuses, releasing
platelets from the tips of the processes into the circulation. Burst- →These are diploid and participate in
→Also found in the lungs Forming normal mitosis, maintaining a viable pool
MEGAKARYOCYTE DIFFERENTIATION AND PROGENITORS Unit (BFU- of megakaryocyte progenitors.
Megakaryocyte Progenitors Meg)
→Arise from the common myeloid progenitor under the Diploid
influence of the transcription gene product, GATA-1 →Paired chromosome
Colony
(Globin Transcription Factor-1), regulated by cofactor Forming
FOG1 (Friend of GATA) →Their proliferative properties are
Unit (CFU-
reflected in their ability to form
Meg)
→Protein product of the X chromosome gene hundreds (BFU-Megs) or scores (CFU-
GATA-1 Megs) of colonies in culture
GATA1
→Product of the ZFMP1 (Zinc Finger Protein →In the third stage, it loses its capacity
FOG1 Light-
Multitype 1) gene. to divide but retains its DNA replication
Density CFU
and cytoplasmic maturation, a partially
(LD-CFU-
Megakaryocyte Differentiation characterized form of mitosis unique to
Meg)
→It is suppressed by another transcription gene product, megakaryocytes known as endomitosis.
MYB (Myeloblastosis), so GATA-1 and MYB act in
opposition to balance megakaryocytopoiesis in one arm ENDOMITOSIS
→A form of mitosis that lacks telophase and cytokinesis
(separation into daughter cells).

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→No cytoplasmic division but has nuclear division.

INVOLVED TRANSCRIPTION FACTORS

→This works as GATA-1 and FOG1
transcription slows down.
→It mediates the switch from mitosis to
endomitosis by suppressing the Rho/ROCK
signaling pathway, which suppresses the
assembly of the actin cytoskeleton.
RUNX1
→In response to the reduced Rho/ROCK
signal, inadequate levels of actin and myosin
(muscle fiber–like molecules) assemble in the
cytoplasmic constrictions where separation
would otherwise occur, preventing
cytokinesis.
→Influences DNA replication that proceeds to
the production of 8N, 16N, or even 32N ploidy
with duplicated chromosome sets.
NF-E2 →Some megakaryocyte nuclei replicate five
times, reaching 128N; this level of ploidy is
unusual, however, and may signal hematologic
disease.

→Megakaryocytes employ their multiple DNA copies to 3 Terminal Megakaryocyte Differentiation Stages
synthesize abundant cytoplasm, which differentiates into
Megakaryoblast
platelets.
→The least differentiated megakaryocyte
precursor
Thrombopoiesis/Thrombocytopoiesis
→No longer look like lymphocytes but it cannot
→Platelet shedding.
be reliably distinguished from bone marrow
→Single Megakaryocyte = Shed 2000 to 4000 platelets
myeloblasts or pronormoblasts (rubriblasts)
→Reference Value for an Average-Size Healthy Human
using light microscopy.
(Total Turnover Rate of 8-9 Days): 108 megakaryocytes
→Has plasma membrane blebs.
producing 1011 platelets per day
→Blebs/Pseudopods: Blunt projections from the
margin that resemble platelets.
→In instances of high platelet consumption, such as
→For locomotion
immune thrombocytopenic purpura, platelet production
→It begins to develop most of its cytoplasmic
rises by as much as tenfold.
structure including:
TERMINAL MEGAKARYOCYTE DIFFERENTIATION o Coagulant-laden α-granules
→As endomitosis proceeds, megakaryocyte progenitors o Dense granules (Dense bodies)
leave the proliferative phase and enter terminal o Demarcation System (DMS)
differentiation
Coagulant- →Their contents and
Terminal Differentiation laden α- functions are described in
→A series of stages in which microscopists become able to granules the subsequent sections on
MK-1
recognize their unique Wright-stained morphology in bone mature platelet
marrow aspirate films or hematoxylin and eosin–stained ultrastructure and function.
bone marrow biopsy sections
Dense granules
α-granules
(Dense bodies)
→For adhesion
→Gray Platelet Syndrome:
Absent α-granules
→A series of membrane-
lined channels that invade
from the plasma membrane
and grow inward to
subdivide the entire
Demarcation
cytoplasm.
System (DMS)
→It is biologically identical
to the megakaryocyte
plasma membrane and
ultimately delineates the
individual platelets during
thrombocytopoiesis.

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→First appears on megakaryocyte progenitors and

! NOTE ! remains present throughout maturation.
*Hallmark: Blebs o CD41: A marker located on the IIb portion
Promegakaryocyte →This is present along with other immunologic markers
→Nuclear lobularity first becomes apparent as an such as:
indentation at the 4N replication stage, rendering o CD36
the cell identifiable by light microscopy. o CD42
MK-II →It reaches its full ploidy level by the end of the o CD61
MK-II stage. o CD62

! NOTE ! Coagulation Factors Detected by Immunostaining in Fully

*Hallmark: Indentation
Megakaryocyte
→The most abundant stage
→Megakaryocyte is easily recognized at 10×
magnification on the basis of its 30- to 50-mm
diameter.
MK-III
→The nucleus is intensely indented or lobulated,
and the degree of lobulation is imprecisely
proportional to ploidy.
→Largest cells and is capable of platelets
shedding.
Mepacrine
→A nucleic acid dye in megakaryocyte flow cytometry to
measure ploidy levels when necessary.
→The chromatin is variably condensed with light and dark
patches.
→The cytoplasm is azurophilic (lavender), granular, and
platelet-like because of the spread of the DMS and α-
granules.
→At full maturation, platelet shedding, or
thrombocytopoiesis, proceeds.
MEGAKARYOCYTE MEMBRANE RECEPTORS AND
MARKERS
Developed Megakaryocyte
→Cytoplasmic Coagulation Factor VIII: Von
1
Willebrand Factor (VWF)
2 →Fibrinogen
THROMBOCYTOPOIESIS: PLATELET SHEDDING
→Platelet shedding.
→One cannot find reliable evidence for platelet budding or
shedding simply by examining megakaryocytes in situ,
even in well structured
bone marrow biopsy preparations.
Megakaryocyte Cultures Examined by Transmission
Electron Microscopy (TEM)
1 →DMS dilates
2 →Longitudinal bundles of tubules form
3 →Proplatelet processes develop
→Transverse constrictions appear throughout the
4
proplatelet processes
→In the bone marrow environment, processes are believed
to pierce through or between sinusoid-lining endothelial
cells, extend into the venous blood, and shed platelets
→Thrombocytopoiesis leaves behind naked
megakaryocyte nuclei to be consumed by marrow
macrophages.
HORMONES AND CYTOKINES OF
MEGAKARYOCYTOPOIESIS

Methods Employed in Specialty and Tertiary Care

Laboratories to Identify Visually Indistinguishable
Megakaryocyte Progenitors in Hematologic Disease
1 →Immunostaining of fixed tissue
2 →Flow Cytometry with immunologic probes Thrombopoietin (TPO)
→Fluorescent in situ hybridization (FISH) with →The growth factor TPO is a 70,000 Dalton molecule that
3 possesses 23% homology with the red blood cell–
genetic probes
producing hormone erythropoietin
Flow Cytometric Megakaryocyte Membrane Markers →Messenger ribonucleic acid (mRNA) for TPO has been
found in the:
→TPO receptor site present in all maturation
MPL o Kidney
stages
o Liver
→Stem cell and common myeloid progenitor
o Stromal cells
CD34 marker
o Smooth muscle cells
→Disappears as differentiation proceeds
o Liver
! NOTE !
Platelet Membrane Glycoprotein IIb/IIIa

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*Liver has the most copies and is considered the primary
source.
→It circulates as a hormone in plasma and is the ligand
that binds the megakaryocyte and platelet membrane
receptor protein identified above, MPL, named for v-mpl
o v-mpl: A viral oncogene associated with murine
myeloproliferative leukemia.
→Plasma Concentration: Inversely proportional to platelet
and megakaryocyte mass, implying that membrane binding
and consequent removal of TPO by platelets is the primary
platelet count control mechanism.
→Investigators have used both in vitro and in vivo
experiments to show that TPO, in synergy with other
cytokines, induces stem cells to differentiate into
megakaryocyte progenitors and that it further induces the
differentiation of megakaryocyte progenitors into
megakaryoblasts and megakaryocytes.
→It also induces the proliferation and maturation
of megakaryocytes and induces thrombocytopoiesis, or
platelet release.
→Synthetic TPO Mimetics (Analogues): Elevate the platelet
count in patients being treated for a variety of cancers,
including acute leukemia.
COMMERCIAL MPL RECEPTOR AGONIST
→ NPlate™, Amgen Inc., Thousand
Oaks, CA, FDA cleared in 2008
→It is a nonimmunogenic
Romimplostim
oligopeptide that is also effective in
raising the platelet count in immune
thrombocytopenic purpura
→ Promacta® and Revolade®, Glaxo
Smith Kline, Inc., Philadelphia, PA,
FDA cleared in 2011
Eltrobopag
→It binds and activates an MPL site
separate from tomiplostim.
→May have additive effects.
CYTOKINES IN SYNERGYST WITH TPO TO STIMULATE
MEGAKARYOCYTOPOIESIS
Interleukin 3 →Induce early differentiation of stem
(IL-3) cells.
Interleukin 6 →Enhance the later phenomena of:
(IL-6) o Endomitosis
o Megakaryocyte maturation
o Thrombocytopoiesis
Interleukin 11 IL-11 Polypeptide Mimetic:
(IL-11) Oprelvekin
→Stimulates platelet production in
patients with chemotherapy-induced
thrombocytopenia.
OTHER CYTOKINES AND HORMONES IN SYNERGYST
WITH TPO AND INTERLEUKINS
→Stem Cell Factor/Kit Ligand/Mast Cell Growth
1 PLATELET COUNT
Factor
→Granulocyte Macrophage Colony Stimulating
2
Factor (GM-CSF)
3 →Granulocyte Colony Stimulating Factor (G-CSF)
→Acetylcholinesterase-derived Megakaryocyte
4
Growth Stimulating Peptide
→Factors that inhibit in vitro megakaryocyte growth,
which means they may have a role in the control of
megakaryopoiesis in vivo:
o Platelet Factor 4 (PF4)
o β-thromboglobulin
o Neutrophil-activating Peptide 2
o IL-8
! NOTE !
*Internally, reduction in the transcription factors FOG1,
GATA-1, and NF-E2 diminish megakaryocytopoiesis at the
progenitor, endomitosis, and terminal maturation process.
PLATELETS
→These are nonnucleated blood cells that circulate at 150-
400 × 109/L, with average platelet counts slightly higher in
women than in men and slightly lower in both sexes when
over 65 years old.
→Metabolically active
→It trigger primary hemostasis upon exposure to
subendothelial collagen or endothelial cell inflammatory
proteins at the time of blood vessel injury.
→On the blood film they have an average diameter of 2.5 mm,
corresponding to a mean platelet volume (MPV) of 8-10 fL
when measured by impedance in a buffered isotonic
suspension, as determined using laboratory profiling
instruments.
→Their internal structure, although complex, is granular but
scarcely visible using light microscopy.
→The proplatelet process sheds platelets, cells consisting of
granular cytoplasm with a membrane but no nucleate
material, into the venous sinus of the bone marrow.
→Circulating, resting platelets are biconvex, although the
platelets in blood collected using the anticoagulant EDTA tend
to round up.
APPEARANCE ON WRIGHT-STAINED WEDGE-
PREPARATION BLOOD FILM
→Platelets are distributed throughout the red blood cell
monolayer at 7-21 cells per 100× field.
→It appears circular to irregular, lavender, and granular,
although their small size makes them hard to examine, for
internal structure.
APPEARANCE IN BLOOD
→Their surface is even, and they flow smoothly
through veins, arteries, and capillaries.
→In contrast to leukocytes, which tend to roll along the
vascular endothelium, platelets cluster with the
erythrocytes near the center of the blood vessel.
→Unlike erythrocytes, however, platelets move back and
forth with the leukocytes from venules into the white pulp
of the spleen, where both become sequestered in dynamic
equilibrium.
REFERENCE VALUES FOR PERIPHERAL BLOOD
Normal →150-400 × 109/L
After 65 Years Men →122-350 × 109/L
Old (Decreased) Women →140-379 × 109/L
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→This count represents only 2/3 of available platelets (8) Platelet Cytoskeleton: Microfilaments and Microtubules
because the spleen sequesters an additional 1/3. (9) Platelet Granules: α-Granules, Dense Granules, and
SEQUESTERED PLATELETS Lysosomes
→These are immediately available in times of demand.
METHODS USED TO STUDY PLATELET ULTRASTRUCTURE
EXAMPLE →Scanning and Transmission Electron
1
→In acute inflammation or after an injury, after Microscopy (SEM and TEM)
1 2 →Flow Cytometry
major surgery, or during plateletpheresis.
→ In hypersplenism or splenomegaly, increased 3 →Molecular Sequencing
2 sequestration may cause a relative RESTING PLATELET PLASMA MEMBRANE
thrombocytopenia. Platelet Plasma Membrane
→Resembles any biological membrane: A bilayer
→Under conditions of hemostatic need, platelets answer composed of proteins and lipids.
cellular and humoral stimuli by:
o Becoming irregular and sticky PREDOMINANT LIPIDS
o Extending pseudopods →Form the basic structure.
o Adhering to neighboring structures or →Form a bilayer with their polar
aggregating with one another heads oriented toward aqueous
RETICULATED PLATELETS environments—toward the plasma
→Sometimes known as stress platelets, appear in externally and the cytoplasm
compensation for thrombocytopenia. internally.
→Markedly larger than ordinary mature circulating →Their fatty acid chains, esterified to
platelets carbons 1 and 2 of the phospholipid
→Their diameter in peripheral blood films exceeds 6 mm, triglyceride backbone, orient toward
and their MPV reaches 12-14 fL. each other, perpendicular to the plane
of the membrane, to form a
APPEARANCE IN TUBES hydrophobic barrier sandwiched
EDTA →Round up within the hydrophilic layers.
Citrated →Cylindrical and beaded, resembling
Whole Blood fragments of megakaryocyte Neutral Phospholipids that
(Blue Top) proplatelet processes. Predominate in the Plasma Layer
→Phosphatidylcholine
→They carry free ribosomes and fragments of rough →Sphingomyelin
endoplasmic reticulum, analogous to RBC reticulocytes,
which triggers speculation that they arise from early and Polar Anionic/Polar Phospholipids that
rapid proplatelet extension and release. Predominates in the Inner Cytoplasmic
Layer
Thiazole Orange →Phosphatidylinositol
Phospholipids
→Nucleic acid dyes such as that bind the RNA of the →Phosphatidylethanolamine
endoplasmic reticulum. →Phosphatidylserine
o This property is exploited by profiling
instruments to provide a quantitative evaluation →These phospholipids, especially
of reticulated platelet production under stress, a phosphatidylinositol,
measurement that may be more useful than the support platelet activation by
MPV. supplying arachidonic acid.
o Platelet dense granules, however, may interfere
with this measurement, falsely raising the Arachidonic Acid
reticulated platelet count by taking up nucleic →An unsaturated fatty acid that
acid dyes. becomes converted to the eicosanoids
prostaglandin and thromboxane A2
→Reticulated platelets are potentially prothrombotic, and during platelet activation.
may be associated with increased risk of cardiovascular
disease. →Phosphatidylserine flips to the outer
surface upon activation and is the
PLATELET ULTRASTRUCTURE charged phospholipid surface on which
(1) Methods Used to Study Platelet Ultrastructure the coagulation enzymes assemble
(2) Resting Platelet Plasma Membrane especially:
(3) Surface-Connected Canalicular System o Coagulation factor complex
(4) Dense Tubular System VIII and IX
(5) Platelet Plasma Membrane Receptors that Provide for o Coagulation factor complex X
Adhesion and V
(6) The Seven-Transmembrane Repeat Receptors →Distributes asymmetrically
Cholesterol
(7) Additional Platelet Membrane Receptors throughout the phospholipids

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→Functions: →The glycocalyx is less developed in the SCCS and lacks

o Stabilizes the membrane some of the glycoprotein receptors present on the platelet
o Maintains Fluidity surface.
o Helps control the →It is the route for endocytosis and for secretion of α-
transmembranous passage of granule contents upon platelet activation.
materials DENSE TUBULAR SYSTEM
Esterified Cholesterol →Parallel and closely aligned to the SCCS
→Moves freely throughout the →It is the control center for platelet activation.
hydrophobic internal layer, exchanging →A condensed remnant of the rough endoplasmic
with unesterified cholesterol from the reticulum.
surrounding plasma. →Having abandoned its usual protein production function
upon platelet release, the DTS sequesters Ca2+ and bears a
Glycoproteins and Proteoglycans series of enzymes that support platelet activation.
→Anchored within the membrane
→These support surface: SERIES OF SETS OF ENZYME SUPPORT
o Glycosaminoglycans 1st Set
o Oligosaccharides →Phospholipase A2
o Glycolipids →Cyclooxygenase
1 →Thromboxane synthetase
Glycocalyx
→The platelet membrane surface also absorbs albumin, →These support the eicosanoid synthesis
fibrinogen, and other plasma proteins, in many instances pathway
transporting them to storage organelles within using a 2nd Set Produced from the Eicosanoid Synthesis
process called endocytosis. 2 →Thromboxane A2
→Phospholipase C
Platelet Glycocalyx 3rd Set Produced from the Support from the 2nd Set
→At 20 to 30 nm, it is thicker than the analogous surface 3 →Inositol triphosphate (IP3)
layer of leukocytes or erythrocytes. →Diacylglycerol (DAG)
→This thick layer is adhesive and responds readily to
hemostatic demands. PLATELET PLASMA MEMBRANE RECEPTORS THAT
→The platelet carries its functional environment with it, PROVIDE FOR ADHESION
meanwhile maintaining a negative surface charge that →The platelet membrane supports more than 50
repels other platelets, other blood cells, and the endothelial categories of receptors, including members of the:
cells that line the blood vessels. o Cell Adhesion Molecule (CAM) Integrin Family
o CAM Leucine-Rich Repeat Family
! NOTE ! o CAM Immunoglobulin Gene Family
*The plasma membrane is selectively permeable o CAM Selectin Family
*Membrane bilayer provides: o Seven-Transmembrane Receptor (STR) Family
→Phospholipids: Support platelet activation internally o Some miscellaneous receptors
→Plasma coagulation: Support platelet activation
externally. Receptors that Support the Initial Phases of Platelet
Adhesion and Aggregation
→The anchored glycoproteins support essential plasma
surface–oriented glycosylated receptors that respond to
cellular and humoral stimuli, called ligands or agonists,
transmitting their stimulus through the membrane to
internal activation organelles.
SURFACE-CONNECTED CANALICULAR SYSTEM
→The plasma membrane invades the platelet interior,
producing its unique surface-connected canalicular system
(SCCS).

Surface-Connected Canalicular System (SCCS)

→Twists spongelike throughout the platelet, enabling the
platelet to store additional quantities of the same
hemostatic proteins found on the glycocalyx and raising its
capacity manyfold.
→It also allows for enhanced interaction of the platelet →Several integrins bind collagen, enabling the platelet to
with its environment, increasing access to the platelet adhere to the injured blood vessel lining.
interior as well as increasing egress of platelet release
products. Integrins

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→Heterodimeric CAMs that integrate their ligands, which cross the platelet
they bind on the outside of the cell, with the internal membrane and
cytoskeleton, triggering activation. interact with actin-
o Heterodimeric: Composed of two dissimilar binding protein to
proteins provide “outside-in”
GLYCOPROTEIN signaling.
ELECTROPHORESIS DESCRIPTION GP IX →Two molecules of
NOMENCLATURE GP IX and one of GP
→An integrin that binds the V help assemble the
subendothelial collagen that four GP Ib molecules.
becomes uncovered in the damaged
blood vessel wall, promoting Bernard-Soulier
adhesion of the platelet to the vessel Syndrome
GP Ia/IIa: Integrin wall. →A moderate-to-
GP V
α2β1 severe
α5β1 and α6β1 mucocutaneous
→Bind the adhesive endothelial cell bleeding disorder
proteins laminin and fibronectin, associated with
which further promotes platelet mutations in GP Iba,
adhesion. GP Ibb, or GP IX (but
→Another collagen-binding not GP V).
receptor that is a member of the
immunoglobulin gene family, so Von Willebrand Factor Defieciency
named because the genes of its →The basis for the most common
GP IV: CAM of the members have multiple inherited bleeding disorder, von
Immunoglobulin immunoglobulin-like domains. Willebrand disease (VWD).
Gene Family →VWD also is associated with
Unclassified Platelet Receptor GP IV mucocutaneous bleeding, although
→A key collagen receptor that also the disorder is technically a plasma
binds the adhesive protein protein (VWF) deficiency, not a
thrombospondin. platelet abnormality.
→An adhesion receptor, a leucine- →The subunits of the integrin GP
rich-repeat family CAM, named for IIb/IIIa are separate and inactive
its members’ multiple leucine rich (αIIb and β3) as they are distributed
domains. across the:
→Arises from the genes: o Plasma membrane
o GP1BA o SCCS
o GP1BB o Internal layer of α-granule
o GP5 membranes.
o GP9
αIIbβ3
COMPOSITION →Active heterodimer
→GP Ibα →Forms to this only when they
Two encounter an “inside-out” signaling
→GP Ibβ
Molecules mechanism triggered by collagen
→GP IX
One → GP V GP IIb/IIIa: Integrin binding to GP VI.
GP Ib/X/V: CAM of αIIbβ3 →Although various agonists may
Molecule
the Leucine-Rich activate the platelet, αIIbβ3 is a
→These total seven noncovalently
Repeat Family physiologic requisite because it
bound subunits.
promotes platelet aggregation
SUBUNITS o Platelet Aggregation:
→Two copies bind Binds fibrinogen,
VWF and support generating interplatelet
platelet tethering cohesion
(deceleration), →Also binds VWF, vitronectin, and
necessary in fibronectin, all adhesive proteins
GP Ibα that share the target arginine-
capillaries and
arterioles where glycine-aspartate (RGD) amino acid
blood flow shear sequence with fibrinogen.
rates exceed 1000 s–
1
. Glanzmann Thrombasthenia
→The accompanying
GP Ibβ
GP Ibβ molecules

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→A severe inherited
mucocutaneous bleeding disorder
caused by mutations in αIIb or β3
THE SEVEN-TRANSMEMBRANE REPEAT RECEPTORS
Platelet Agonists that Function Individually or Together to
Activate Platelets
1 →Thrombin
2 →Thrombin Receptor Activation Peptide (TRAP)
3 →Adenosine Diphosphate (ADP)
4 →Epinephrine
→Eicosanoid synthesis/ Prostaglandin/
5 Cyclooxygenase Pathway product Thromboxane
A2 (TXA2)
→These platelet “agonists” are ligands for the seven-
transmembrane repeat receptors (STRs), so named for
their
unique membrane-anchoring structure.
Seven-Transmembrane Repeat Receptors (STRs)
→They have seven hydrophobic anchoring domains
supporting an external binding site and an internal
terminus that interacts with G proteins for outside-in
platelet signaling.
LIGAND DESCRIPTION
→It cleaves two STRs, protease-activated
receptor 1 (PAR1) and PAR4, that
together have a total of 1800 membrane
copies on an average platelet.
→Thrombin cleavage of either of these
two receptors activates the platelet
through G-proteins that in turn activate
Thrombin
at least two internal physiologic
pathways, described subsequently.
→It also interacts with platelets by
binding or digesting two CAMs in the
leucine-rich repeat family, GP Iba and GP
V, both of which are parts of the GP
Ib/IX/V VWF adhesion receptor.
→There are about 600 copies of the
ADP high-affinity ADP receptors P2Y1 and
P2Y12 per platelet.
→These STRs also activate the platelet
through the G-protein signaling
pathways.
→TPa and TPb bind TXA2.
→This interaction produces more TXA2
Thromboxane
from the platelet, a G-protein based
A2 (TXA2)
autocrine (self-perpetuating) system that
activates neighboring platelets.
→Binds α2-adrenergic sites that also
couple to G-proteins and open up
membrane calcium channels.
Epinephrine
α2-adrenergic Sites
→Function similarly to those located on
heart muscle.
→Receptor site IP binds to this.
→A prostaglandin produced from
endothelial cells.
Prostacyclin/
→Prostacyclin enters the platelet and
Prostaglandin
raises the internal cyclic adenosine
I2 (PGI2)
monophosphate (cAMP) concentration of
the platelet, thus blocking platelet
activation.
→The platelet membrane also presents STRs for:
o Serotonin
o Platelet-activating factor
o Prostaglandin E2
o PF4
o β-thromboglobulin
ADDITIONAL PLATELET MEMBRANE RECEPTORS
→About 15 clinically relevant receptors were discussed in
the preceding paragraphs.
→The platelet supports many additional receptors.
CAM IMMUNOGLOBULIN FAMILY
CD50, CD54, and CD102
ICAMs →Play a role in inflammation and the immune
reaction
CD31
PECAM →Mediates platelet–to–white blood cell and
platelet–to–endothelial cell adhesion
CD32
→A low-affinity receptor for the
FcγIIA immunoglobulin Fc portion that plays a role
in a dangerous condition called heparin-
induced thrombocytopenia.
P-selectin (CD62)
→An integrin that facilitates platelet binding to endothelial
cells, leukocytes, and one another.
→Found on the α-granule membranes of the resting
platelet but migrates via the SCCS to the surface of
activated platelets.
→P-selectin or CD62 quantification by flow cytometry is a
successful clinical means for measuring in vivo platelet
activation.
PLATELET CYTOSKELETON: MICROFILAMENTS AND
MICROTUBULES
→A thick circumferential bundle of microtubules maintains
the platelet’s discoid shape.
! NOTE !
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*Microtubules, actin microfilaments, and intermediate

microfilaments control platelet shape change, extension of
pseudopods, and secretion of granule contents.
→It parallels the plane of the outer
surface of the platelet and reside just
within, although not touching, the
plasma membrane.
→There are 8-20 tubules composed of
multiple subunits of tubulin that
disassemble at refrigerator
temperature or when treated with
colchicine.

Microtubules that Disassemble in the

Cold
→Platelets become round, but upon
Circumferential
warming to 37° C, they recover their
Microtubules
original disc shape.
α-Granule
→On cross section, microtubules are
cylindrical, with a diameter of 25 nm.
→The circumferential microtubules →There are 50-80 α-granules in each
could be a single spiral tubule. platelet.
→Besides maintaining the platelet →Unlike the nearly opaque dense granules,
shape, microtubules move inward on α-granules stain medium gray in osmium-
activation to enable the expression of dye transmission electron microscopy
α-granule contents. preparations.
→They also reassemble in long parallel →They are filled with proteins, some
bundles during platelet shape change endocytosed, some synthesized within the
to provide rigidity to pseudopods. megakaryocyte and stored in platelets.
→In the narrow area between the →Several α-granule proteins are
microtubules and the membrane lies a membrane bound.
thick meshwork of microfilaments →As the platelet becomes activated, α-
composed of actin. granule membranes fuse with the SCCS.
→It is contractile in platelets (as in →Their contents flow to the nearby
muscle) and anchors the plasma microenvironment, where they participate
membrane glycoproteins and in platelet adhesion and aggregation and
proteoglycans. support plasma coagulation.
→It also is present throughout the
platelet cytoplasm, constituting 20%-
Actin 30% of platelet protein.

APPEARANCE
→Globular and
Resting Platelet
amorphous
Rise in →Filamentous
Cytoplasmic and contractile.
Calcium
Concentration Dense
Granules/ →There are 2-7 dense granules per
→Ropelike polymers 8-12 nm in Dense platelet.
diameter, of desmin and vimentin. Bodies →These granules appear later than α-
Intermediate granules in megakaryocyte differentiation
→The intermediate filaments connect
Filaments and stain black (opaque) when treated with
with actin and the tubules, maintaining
the platelet shape. osmium in transmission electron
PLATELET GRANULES: α-GRANULES, DENSE GRANULES, microscopy.
AND LYSOSOMES →Small molecules are probably
endocytosed and are stored in the dense
PLATELET
DESCRIPTION granules.
GRANULE
→In contrast to the α-granules, which
employ the SCCS, dense granules migrate to
the plasma membrane and release their

9
1

contents directly into the plasma upon ADAMTS-13/VWF-Cleaving Protease

platelet activation. →A liver-secreted plasma enzyme that keep platelet and
→Membranes of dense granules support VWF interactions remain localized
the same integral proteins as the α- →Digests “unused” VWF.
granules which implies a common source
for the membranes of both types of →At high shear rates, the VWF-GP Ibα tethering reaction is
granules: temporary, and the platelet rolls along the surface unless
o P-selectin GP VI comes in contact with the exposed ECM collagen.
o αIIbβ3
o GP Ib/IX/V Internal Platelet Activation Pathway: Outside-In
→Platelets also have a few lysosomes, Reaction
similar to those in neutrophils, 300-nm- →When type I fibrillar collagen binds platelet GP
diameter granules VI, the receptor, which is anchored in the
→Stain positive for: membrane by an Fc receptor–like molecule,
o Arylsulfatase triggers internal platelet activation pathways,
1
Lysosomes o β-glucuronidase releasing TXA2 and ADP, an “outside-in” reaction.
o Acid phosphatase
o Catalase Outside-In Reaction Summary
→These probably digest vessel wall matrix Type I Fibrillar Collagen + GP VI (Receptor) =
components during in vivo aggregation and Internal Platelet Activation Pathways = TXA2 and
may also digest autophagic debris. ADP (Both agonist)
Raised Affinity of Integrin α2β1: Inside-Out
PLATELET ACTIVATION Reaction
(1) Adhesion: Platelets Bind Elements of the Vascular Matrix →These agonists attach to their respective
(2) Aggregation: Platelets Irreversibly Cohere receptors:
(3) Secretion: Activated Platelets Release Granular Contents AGONIST RECEPTOR
2 →TPα
→A linear and stepwise process. TXA2
→TPβ
ADHESION: PLATELETS BIND ELEMENTS OF THE →P2Y1
VASCULAR MATRIX ADP
→P2Y12
→As blood flows, vessel walls create stress, or shear force, →This triggers an “inside-out” reaction that raises
measured in units labeled s-1. the affinity of integrin α2β1 for collagen.
Firm Attachment of Platelet to the Damaged
RANGES OF SHEAR FORCE Surface
Venules and Veins →500 s-1 →The combined effect of GP Ib/IX/V, GP VI, and
3
Arterioles and Capillaries →5000 s-1 α2β1 causes the platelet to become firmly affixed
Stenosed/Hardened Arteries →40,000 s-1 to the damaged surface, where it subsequently
loses its discoid shape and spreads.
→In vessels where the shear rate is over 1000 s-1, platelet
adhesion and aggregation require a defined sequence of Internal Platelet Activators: TXA2 and ADP
events that involves: →These are also secreted from the platelet to the
o Collagen microenvironment, where they activate neighboring
o Tissue Factor platelets through their respective receptors.
o Phospholipid →Further, they provide inside-out activation of integrin
o VWF αIIbβ3, the key receptor site for fibrinogen, which assists in
o A number of platelet CAMs, ligands, and activators platelet aggregation.
→Injury to the blood vessel wall disrupts the collagen of AGGREGATION: PLATELETS IRREVERSIBLY COHERE
the →In addition to collagen exposure and VWF secretion,
extracellular matrix (ECM). blood vessel injury releases constitutive (integral) tissue
→Damaged endothelial cells release VWF from cytoplasmic factor from endothelial cells.
storage organelles.
Tissue Factor
Von Willebrand Factors 1 →Triggers the production of thrombin, which
→Has a molecular weight ranges from 800,000-2,000,000 reacts with platelet STRs PAR1 and PAR4.
Daltons. →This further activation generates the “collagen
→It unrolls like a carpet and adheres to the injured site. and thrombin activated” or COAT platelet, integral
→Though VWF circulates as a globular protein, it become 2
to the cell-based coagulation model described in
fibrillar as it unrolls and exposes sites that partially bind Chapter 37 (Figure 13-16).
the GP Ibα portion of the platelet membrane GP Ib/IX/V →Meanwhile, integrin αIIbβ3 assembles from its
leucine-rich receptor. resting membrane units αIIb and β3, binding RGD
→This is a reversible binding process that tethers or 3
sequences of fibrinogen and vWF and supports
decelerates the platelet. platelet-to-platelet aggregation.

10
1

→P-selectin from the α-granule membranes DESIGNATION OF GRANULAR CONTENTS

4 moves to the surface membrane to further α-Granules and →Contents of flow through the
promote aggregation. Lysosomes SCCS
→Platelets lose their shape and extend →Migrate to the plasma
5
pseudopods. Dense Granules membrane, where their contents
→Membrane phospholipids redeploy with the are secreted.
more polar molecules, especially
6 phosphatidylserine, flipping to the outer layer, Dense Granular Contents
establishing a surface for the assembly of →These are vasoconstrictors and platelet agonists that
coagulation factor complexes. amplify primary hemostasis
→As platelet aggregation continues, membrane →Most of the α-granule contents are coagulation proteins
integrity is lost, and a syncytium of platelet that participate in secondary hemostasis.
7
cytoplasm forms as the platelets exhaust internal
energy sources. ! NOTE !
*Dense Granules: Primary Hemostasis
Platelet Aggregation *α-Granules: Secondary Hemostasis
→A key part of primary hemostasis, which in arteries may
end with the formation of a white clot, →By presenting polar phospholipids on their membrane
surfaces, platelets provide a localized cellular milieu that
White Clot supports coagulation.
→A clot composed primarily of platelets and VWF.
→Although aggregation is a normal part of vessel repair, Phosphatidylserine
white clots often imply inappropriate platelet activation in →The polar phospholipid on which two coagulation
seemingly uninjured arterioles and arteries and are the pathway complexes assemble:
pathological basis for arterial thrombotic events, such as: o Tenase: Factor IX/VIII
o Acute myocardial infarction o Prothrombinase: Factor X/V
o Peripheral artery disease
o Strokes ROLE OF GRANULAR CONTENTS IN TENASE AND
→The risk of these cardiovascular events rises in PROTHROMBINASE
proportion to the numbers and avidity of platelet Dense Granules →Secretes ionic calcium that
membrane α2β1 and GP VI. supports both factors.
→Scretes the following to
Coagulation: Secondary Hemostasis increase the localized
→Triggered by the combination of: concentrations of these essential
o Polar phospholipid exposure on activated coagulation proteins:
platelets α-Granules o Fibrinogen
o Platelet fragmentation with cellular microparticle o Factors V and VIII
dispersion o VWF
o Secretion of the platelet’s a-granule and dense →VWF binds and stabilizes factor
granule contents VIII
→Their presence further supports the action of tenase and
→Fibrin and RBCs deposit around and within the platelet prothrombinase.
syncytium, forming a bulky “red clot”.
Platelet secretions
Red Clot →Provide for cell-based, controlled, localized coagulation.
→Essential to wound repair, but it may also be
characteristic of inappropriate coagulation in venules and
veins, resulting in deep vein thrombosis and pulmonary
emboli.
SECRETION: ACTIVATED PLATELETS RELEASE
GRANULAR CONTENTS

COMBINED TRIGGERS FOR ACTIN MICROFILAMENT

CONTRACTION
1 →Outside-in platelet activation through ligand
(agonist) binding to integrins
2 → STRs (such as ADP binding to P2Y12)
3 →Immunoglobulin gene product GP VI →This lists some additional α-granule secretion products
that, although not proteins of the coagulation pathway,
→Intermediate filaments also contract, moving the indirectly support hemostasis.
circumferential microtubules inward and compressing the →The lists in Tables 13-6, 13-7, and 13-8 are not
granules. exhaustive because more and more platelet granule

11
1
contents continue to be identified through platelet research
activities.
PLATELET ACTIVATION PATHWAYS
(1) G Proteins
(2) Eicosanoid Synthesis
(3) Inositol Triphosphate–Diacylglycerol Activation Pathway
G PROTEINS
→Control cellular activation for all cells (not just platelets)
at the inner membrane surface.
→G proteins are αβγ heterotrimers that bind guanosine
diphosphate (GDP) when inactive.
o αβγ Heterotrimers: Proteins composed of three
dissimilar peptides
→Membrane receptor-ligand (agonist) binding promotes
GDP release and its replacement with guanosine
triphosphate (GTP).
→The Gα portion of the three-part G molecule briefly
disassociates, exerts enzymatic guanosine triphosphatase
activity, and hydrolyzes the bound GTP to GDP, releasing a
phosphate radical.
→The G protein resumes its resting state, but the
hydrolysis step provides the necessary phosphorylation
trigger to energize the eicosanoid synthesis or the IP3-DAG
pathway (Table 13-9).
EICOSANOID SYNTHESIS/IP3-DAG PATHWAY
→The eicosanoid synthesis pathway, alternatively called
the prostaglandin, cyclooxygenase, or thromboxane
pathway, is one of two essential platelet activation
pathways triggered by G proteins
→The platelet membrane’s inner leaflet is rich in
phosphatidylinositol
Phosphatidylinositol
→A phospholipid whose number 2 carbon binds numerous
types of unsaturated fatty acids, but especially 5,8,11,14-
eicosatetraenoic acid, commonly called arachidonic acid.
→Membrane receptor-ligand binding and the consequent
G-protein activation triggers phospholipase A2
Phospholipase A2
→A membrane enzyme that cleaves the ester bond
connecting the number 2 carbon of the triglyceride
backbone with arachidonic acid.
→Cleavage releases arachidonic acid to the cytoplasm,
where it becomes the substrate for cyclooxygenase,
anchored in the DTS.
Cyclooxygenase
→Converts arachidonic acid to prostaglandin G2 and
prostaglandin H2,
Thromboxane Synthetase
→Acts on prostaglandin H2 to produce TXA2.
→TXA2 binds membrane receptors TPα or TPβ,
decelerating adenylate cyclase activity and reducing cAMP
concentrations, which mobilizes ionic calcium from the
DTS.
→The rising cytoplasmic calcium level causes contraction
of actin microfilaments and platelet activation.
Cyclooxygenase Pathway in Endothelial Cells
→Incorporates the enzyme prostacyclin synthetase in
place of the thromboxane synthetase in platelets.
→The eicosanoid pathway end point for the endothelial
cell is prostaglandin I2/prostacyclin, which infiltrates the
platelet and binds its IP receptor site.
→Prostacyclin binding accelerates adenylate cyclase,
increasing cAMP, and sequesters ionic calcium to the DTS.
→The endothelial cell pathway suppresses platelet
activation in the intact blood vessel through this
mechanism, creating a dynamic equilibrium.
→TXA2 has a half-life of 30 seconds, diffuses from the
platelet, and becomes spontaneously reduced to
thromboxane B2,
o Thromboxane B2: A stable, measurable plasma
metabolite.
→Efforts to produce a clinical assay for plasma
thromboxane B2 have been unsuccessful, because special
specimen management is required to prevent ex vivo
platelet activation with unregulated release of
thromboxane B2 subsequent to collection.
→Thromboxane B2 is acted on by a variety of liver enzymes
to produce an array of soluble urine metabolites, including
11-dehydrothromboxane B2, which is stable and
measurable
INOSITOL TRIPHOSPHATE–DIACYLGLYCEROL
ACTIVATION PATHWAY
→The IP3-DAG pathway is the second G protein–dependent
platelet activation pathway.
→G-protein activation triggers the enzyme phospholipase
C.
Phospholipase C
→Cleaves membrane phosphatidylinositol 4,5-
bisphosphate to form IP3 and DAG, both second
messengers for intracellular activation.
→Promotes release of ionic calcium from the
DTS, which triggers actin microfilament
IP3
contraction.
→May also activate phospholipase A2.
→Triggers a multistep process: Activation of
phosphokinase C, which triggers
DAG
phosphorylation of the protein pleckstrin, which
regulates actin microfilament contraction.
Second Messengers
→Internal platelet activation pathways, like internal
pathways of all metabolically active cells,
12
1

→They are triggered by a primary ligand-receptor binding →As the cell enters endomitosis, this signifies the
event. 3 end of proliferative stage and starts to enter the
→This include: terminal megakaryocyte differentiation.
o G proteins
o Eicosanoid Synthesis Pathway TERMINAL MEGAKARYOCYTE DIFFERENTIATION
o IP3-DAG Pathway →This involves the 3 terminal megakaryocyte
o Adenylate Cyclase differentiation stages:
o cAMP o MK-I (Megakaryoblast)
o Intracellular Ionic Calcium o MK-II (Promegakaryocyte)
o MK-III (Megakaryocyte)

Synchronous Discussion →MK-I plus the thrombopoietin (TPO)

Thrombopoietin
TOPIC OUTLINE →It is a growth factor that helps the stimulation
1 Platelet Production and proliferation of the platelets
2 Platelet Structure 1 →This is needed from MK-I to MK-III
3 Platelet Function
4 History MK-I
→Least differentiated megakaryocyte
PLATELET PRODUCTION →Has blebs that is responsible for locomotion
→Hallmark: Presence of blebs
MK-II
2
→Hallmark: Presence of indentation or cleavage
MK-III
→Most abundant and easily recognized.
3 →It is capable of platelet shedding that is involved
in the last stage of platelet production,
thrombopoiesis stage.

! NOTE !
*MK-I, MK-II, and MK-III is no longer capable of mitosis
THROMBOPOIESIS/PLATELET SHEDDING STAGE
Platelet Shedding
→1 Megakaryocyte = 2000-4000 Platelets

FEATURES OF TERMINAL MEGAKARYOCYTE

Multipotential Hematopoietic Stem Cell DIFFERENTIATION
→Divided into 2 progenitors:
o Common Myeloid Progenitor (CMP)
• Erythropoiesis
• Granulopoiesis
• Megakaryopoiesis
o Common Lymphoid Progenitor (CLP)
• Lymphocyte Production
→Progenitors are undifferentiated or immature
hematopoietic cell that is committed to a cell line
PRODUCTION OF PLATLETS: PLATELET MATURAITON
PROLIFERATIVE STAGE
→From the CMP arise 3 megakaryocyte lineage committed
progenitors:
o BFU-Meg
o CFU-Meg
o LD-CFU-Meg
! NOTE !
*All of these resembles lymphocytes.

→BFU-Meg and CFU-Meg are diploid and are

1 capable of mitosis giving them their proliferative
ability.
→LD-CFU-Meg is no longer capable of mitosis but
2
is able to progress to endomitosis. ! NOTE !

13
1

*All of the stages contain the α-granules, dense granules,

demarcation system (DMS).

Precursors
→Immature hematopoietic cell that is morphologically
identifiable
THROMBOPOIETIN
→A glycoprotein hormone produced mainly by the liver
and the kidney that regulates the production of platelets by
the bone marrow.
→It stimulates the production and differentiation of
megakaryocytes.
MEGAKARYOCYTE
→Bone marrow cell which is responsible for the
production of matured platelets.
→Accounts for about 1% of total nucleated cells in the
bone marrow. THROMBOCYTE/PLATELET
→The only cell line where cells become bigger as they →Nonnucleated cells (Anucleate).
mature. →Disk-shaped cell arise from megakaryocyte.
→Do not undergo mitosis →1/3 (30%) are sequestered in the spleen
→Do not increase in number as they mature. →70% are in the circulation
MEGAKARYOCYTE MEMBRANE RECEPTORS AND MARKER →Reference Value: 150-400 × 109/L
→Life Span: 8-12 Days
→Platelet Satellitism: A neutrophil or monocyte
surrounded by platelets due to anticoagulants.
→Platelets are responsible for primary hemostasis
→Coagulation factors are involved in secondary
hemostasis
→Diameter: 2.5 µm
→Mean Platelet Volume (MPV): 8-10 fL
o Increased MPV signifies large platelets that is not
good and may cause Bernard-Soulier Syndrome
(BSS)
→Shape: Disk-shaped or circular to irregular, lavender and
Fibrinogen/FI granular under Wright-stained wedge preparation
→Its function is for adhesion
HORMONES AND CYTOKINES THAT CONTROLS PLATELET STRUCTURE
MEGAKARYOPOIESIS (1) Peripheral Zone
(a) Glycocalyx
(b) Plasma Membrane
o Glycoprotein Ib (GP Ib)
o Glycoprotein IIb-IIIa (GP IIb-IIIa)
(2) Sol-Gel Zone
(a) Microtubules
(b) Microfilaments
(3) Organelle Zone/Centromere
(a) α-Granules
(b) Dense Granules/Delta
(c) Mitochondria
(d) Lysosomal Granules
(4) Membranous System
→Produciton of platelets must be balanced because it can (a) Dense Tubular System (DTS)
affect the coagulation mechanism. (b) Open Canalicular System (OCS)

Hormones and Cytokines

→These are growth factors that will help in the production
of platelets.
MEGAKARYOPOIESIS

14
1

→It gives the basic structure of the

platelet other than microtubules and
microfilaments.

Cholesterol
→Distributed throughout the
phospholipids

2 GLYCOPROTEIN OF PLASMA
MEMBRANE
→Serves as the
binding site for
vWF (von
Willebrand
Factor),
necessary for
platelet adhesion.
Glycoprotein
A: Peripheral Zone Ib (GP Ib)
Von Willebrand
B: Organelle Zone/Centromere
Factor
C: Structural Zone
→Helps in
D: Membranous System
adhesion
→Absence can
MAJOR STRUCTURAL FEATURE OF PLATELET
cause a lot of
→Composed of the membranes and is responsible for bleeding.
platelet adhesion and aggregation
→Calcium-
→Originates from the plasma membrane of the
dependent
megakaryocytes
membrane
Platelet Adhesion and Aggregation
Glycoprotein protein complex
→Adhesion of platelet to a non-platelet
IIb-IIIa (GP for fibrinogen
→In an open wound, platelet will be the first to go at
IIb-IIIa) receptor
the site of injury that indicates platelet adhesion
necessary for
(platelet to non-platelet)
platelet
→After platelet adhesion, it will further clump that
aggregation.
indicates platelet aggregation (platelet to platelet).

! NOTE !
→Lies directly beneath the platelet membrane.
*Platelet Adhesion: Platelet to Non-Platelet
→The structural zone that gives the platelet its disc-
*Platelet Aggregation: Platelet to Platelet
shape.
2 DIVISIONS
SOL-GEL ZONE

→Composed of protein tubulin

→Primarily composed of glycoproteins
PERIPHERAL ZONE

Microtubules which maintains the platelet disc

including:
shape.
o Factor V: Heat Labile Factor
→Contain actin and myosin which
o Factor VIII: Leiden Factor
upon stimulation of the platelet
o Factor I: Fibrinogen
Microfilaments will it react to form actomyosin
Glycocalyx →The glycoprotein factors are
(thrombosthenin) for clot
responsible for adhesion (FV and FVIII)
retraction
and aggregation (FI).
→It is full of platelet receptors →The part where the secretory products of platelet
come from.
ORGANELLE ZONE/ CENTROMERE

Platelet Receptors →Main Function: Adhesion

→Serves as the physical and chemical
barrier between the intracellular and ! NOTE !
extracellular constituents of the 1 Platelt = 50-80 α-granules
platelets. →Composed of a bilayer of Platelet Factor → Anti-heparin
asymmetrically distributed 4
phospholipids imbedded with integral →Promote
Plasma α-Granules
surface receptors. smooth muscle
Membrane growth for
→Also responsible for adhesion and
aggregation. β-thrombo- vessel repair
→The predominant lipid is globulin
phospholipid ! NOTE !
*Coagulation or
Phospholipids hemostasis, it

15
1

will involve Acid →Digests the

blood vessel Phosphatase materials that is
repair in which Lysosomal brought by the
the culprit is the Granules Hydrolytic platelet by
injury. Enzymes endocytosis
→Also →For lyses
Platelet-
promotes →Derived from smooth ER and
derived Growth
smooth muscle sequesters calcium for platelet
Factor (PDGF)
growth activation process.
Fibrinogen →For adhesion →It is also the site of platelet
Dense Tubular
Factor V cyclooxygenase and of
System
(Leiden Factor) prostaglandin synthesis.
vWF
→Promote Cyclooxygenase and Prostaglandin
Thrombo- platelet-to- →Important for aggregation
spondin platelet →Surface Connecting System

MEMBRANOUS SYSTEM
interaction. →An invagination of the plasma
membrane
Fibronectin →Acts as a canal for the release of
! NOTE ! the granule constituents and
1 Platelet = 2-7 Dense Granules cytoplasm to the exterior of the
platelet.
→Involved in
→This is where all of the platelet
ADP platelet
Open contents pass through once
aggregation
Canalicular platelet activation or
→Energy source
ATP System release/secretion mechanism
of platelet
occurs.
→Important in
the activation of
Platelet Activation/Release
coagulation
Mechanism
cascade
→Platelet will now have
(activation of
protrusions and will loose its disc-
coagulation
shape structure and will form
factors)
pseudopods.
→Without it,
bleeding will not
PLATELET FUNCTION
stop
→Participates in a sequence of events that lead to the
formation of a platelet plug and ultimately to the formation of
EXAMPLE
a stable fibrin clot at the site of vessel interruption.
Dense →Fibrinogen
Granules/ Delta (FI) is an Stable Fibrin Clot→Platelet Plug/Hemostatic Platelet Plug
inactive →After platelet adhesion and aggregation, it will temporary
Calcium and coagulation stop the bleeding.
Magnesium factor that will →Fibrin will form at the site of injury that will led to the
be activated by formation of platelet plug.
thrombin to →Once fibrin is formed, bleeding will no longer be temporary
form fibrin or since it will serve as the cement to stop the bleeding.
FIa (active →Fibrin will be discussed in the coagulation pathway
form)
Primary Hemostasis
1 →By adhesion, secretion and aggregation with the
! NOTE ! view of hemostatic plug.
*In writing
Blood Coagulation
coagulation
→By releasing platelet factor 3 that plays a big role in
factors, it should
forming fibrin clot.
always be in
2
Roman Numerals
! NOTE !
and addition of
* Thrombin is also important because it activates
“a” indicates that
fibrinogen to form fibrin
it is activated.
Clot Retraction
Serotonin →For constriction 3
→By its actomyosin
→For ATP synthesis used for
Mitochondria →Helps in localization of bacteria and other small
platelet metabolism
4 objects and producing aggregates too large to pass
through capillaries.

16
1

PLATELET ROLE ON HEMOSTASIS →Lee and White Test

PLATELET Quick
ACTIVITY
FACTORS →Introduced prothrombin time
→Accelerates the conversion of PT to 1930
PF1
thrombin Prothrombin Time Reference Value
→Accelerates the clotting of purified →10-15 seconds
PF2
fibrinogen by thrombin →Other tests for evaluating hemostatic
→Phospholipid needed in the intrinsic mechanisms were introduced like:
PF3
coagulation pathway
→Anti-heparin Platelet →Uses RBC
PF4 →Part of α-granules that neutralizes Count Direct pipette and
heparin. 1940 hemocytometer
PF5 →Necessary for normal fibrin formation →Uses blood
Indirect
→Anti-fibrinolysis smear
Bleeding
Fibrinolysis Time (BT)
PF6
→Removes the platelet plug once the
bleeding stop and healed to open the →Cascade and Waterfall Theory of
endothelium/endothelial cells once again. 1964
Coagulation was introduced.
→Necessary in the formation of intrinsic
PF7
thromboplastin (FIII)
! NOTE !
*There are 3 Coagulation Factor (ICE)
→Intrinsic
→Common
→Extrinsic

HISTORY

→Hemophilia was first recognized

→Hemophilia A, B, and C

Hemophilia
2nd Century →Absence of coagulation factors
A.D. Hemophilia A →FVIII: Leiden Factor
Hemophilia B →F IX: Christmas Factor
→FXI: Plasma
Hemophilia C
Thromboplastin

Moises Maimonides
→Described 2 male siblings who died
12th
because of excessive bleeding after
Century
circumcision.
A.D.
→Excessive bleeding may be caused by
problems with coagulation factors
→Clinical description of families with
hemophilia was first published.

1803 Schonlein
→Gave the disorder was given the name
hemophilia which means “love of
hemorrhage”.
Hoff
1828 →The disorder was first described in his
thesis
1842 →Platelets were described.
Paul Morawitz
1905 →His theory was accepted which is Theory
on Blood Coagulation
Lee and White
1913 →WBCT(Whole Blood Clotting Test) was
performed

17
1

PRINCIPLE OF COAGULATION →von Willebrand Factor (vWF) and

GP Ib is needed for platelet
Platelet Adhesion
TOPIC OUTLINE adhesion.
1 Hemostasis →vWF + GP Ib
2 Vasoconstriction →Fibrinogen, vWF, and GP IIb/IIIa
3 Platelet Adhesion and Aggregation Platelet (Integrin αIIbβ3) initiates binding of
4 Arteries and Veins Aggregation platelet to platelet.
5 Role of Blood Vessels in Hemostasis →Fibrinogen + vWF + GP IIb/IIIa
6 The Key Cellular Elements of Hemostasis
7 Role of Coagulation in Hemostasis ARTERIES AND VEINS
8 Concept of Normal Coagulation
9 Concept of Hypocoagulation
10 Concept of Hypercoagulation

HEMOSTASIS
→Hemo: Means blood; Stasis: Means stoppage
→Involves the interaction of vasoconstriction, platelet
adhesion and aggregation and coagulation enzymes
→A complex process that:
o Produces a clot to stop the bleeding
• Involvement of primary and secondary
hemostasis
o Keeps the clot confined
• Involvement of fibrinogen (FI) to be
activated to fibrin (FIa)
• Fibrin will support the clot made by
primary hemostasis
o Dissolves the clot as the wound heals
• Involvement of fibrinolysis
• Fibrinolysis will destroy the fibrin to go
back to its normal state
▪ This involves plasmin inhibitors
and activators
→It also includes the coagulation enzyme
VASOCONSTRICTION
→Reduction in blood vessel diameter due to smooth muscle
constriction
→This occurs in cases of blood vessel injury
Arteries →Have the thickest wall of the vascular system
→Larger and have more irregular lumen than
Veins
arteries
→Blood vessel is coated into 3 layers (IMA):
→The inner lining/innermost layer
Tunica intima
→Composed of endothelial cells
→The 2nd layer
Tunica media →Composed of smooth muscle and
elastic tissue
→Outermost layer
→Shallow injury only involves
primary hemostasis
Tunica adventitia
→Deep injury involves secondary
hemostasis that involves the
coagulation factors those are platelets
ROLE OF BLOOD VESSELS IN HEMOSTASIS
→Blood flows through the vascular system to and from all
parts of the body.
→The vascular system consists of (CAV):
o Capillaries
o Arteries
o Veins
→Blood normally carried within vessels whose physical
capabilities include contraction (vasoconstriction/
narrowing) and dilation, which are controlled by the
smooth muscle of the vessel media.

PLATELET ADHESION AND AGGREGATION

→Responsible for microcirculation that links

between arterial and venous circulation.
→Thinnest walled and most numerous of
Capillaries blood vessels compose of basement
membrane that is tightly anchored with
collagen that offers support for microvascular
unit.
→ Smaller blood vessels

18
1

→Microscopic continuation of →Inhibits or slows down the

Prostacyclin
Arterioles Arterioles arteries giving off metaarterioles platelet activation and aggregation.
and joining capillaries →Enhances activity of
Venules →Microscopic size of veins antithrombin III (AT III)
Venules
connecting to capillaries
! NOTE ! Antithrombin III
*Hemostasis start with the culprit which is the vessel injury →Inactivate thrombin
→Thrombin activates fibrinogen to
Heparan Sulfate
THE KEY CELLULAR ELEMENTS OF HEMOSTASIS form fibrin

1 →Vascular intima →Inhibition prevents the

2 →Extravascular tissue factor (TF/ Factor III) disruption of balance
3 →Platelets →Prevents bleeding and
4 →Blood Coagulation Factors thrombosis
Fibrinolytic Proteins Tissue Factor →Anticoagulants produced by
→These are involved in fibrinolysis Pathway endothelial cells
5 Inhibitor (TFPI)/ →Controls the activation of tissue
→This includes the plasmin and tissue plasminogen
activators Extrinsic factors pathway
VASCULAR INTIMA Coagulation
Pathway
→Induces smooth muscle
Nitric Oxide relaxation (vasodilation)
→Inhibits platelet activation
→Present in the endothelial cells
Thrombomodulin
that acts as a normal anticoagulant.
! NOTE !
→The vascular intima provides the interface between *Activation and inactivation maintains balance to prevent
circulating blood and the body tissues. bleeding
→The innermost lining of blood vessels is a monolayer of
metabolically active endothelial cells VASCULAR INTIMA OF THE BLOOD VESSELS
Innermost →Endothelial cells (endothelium)
ROLE OF ENDOTHELIAL CELLS Vascular Lining
Immune Response Supporting the →Internal elastic lamina composed
1 Endothelial Cells of elastin and collagen
→It is an essential part of the immune system
Vascular Permeability →Collagen and
Veins
2 →Capacity of the vessel wall to allow the flow of Subendothelial fibroblasts
the blood. Tissue →Collage, fibroblasts,
Arteries
Proliferation and smooth muscle cells
3 →Promotes hematopoietic stem cell (HSC) for EXTRAVASCULAR COMPONENT
proliferation and differentiation →The extra-vascular (tissues surrounding blood vessels)
Hemostasis involved in hemostasis when local vessel is injured,
4 →Maintain the survival and self-renewal of through swelling and inflammation
hematopoietic stem cells (HSC) →It plays a part in hemostasis by providing back-pressure
on the injured vessel through swelling and trapping of
Normal Blood Flow in Intact Vessels escaped blood.
PLATELETS
→The platelets and plasma proteins that circulate within
the blood vessels.
→These components are involved in coagulation or
fibrinolysis

Coagulation →Clot or thrombus formation

→Clot or thrombus dissolution
Fibrinolysis →Destruction/Lysis when there is
wound healing

→Once endothelial cells is broken, it will release the

following to promote adhesion: ROLE OF COAGULATION IN HEMOSTASIS
o Tissue Factor 3 (FIII)
o von Willebrand Factor (vWF) Coagulation
COMPONENTS NEEDED FOR NORMAL BLOOD FLOW IN
INTACT VESSELS

19
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→It is the process whereby on vessel injury, plasma →Deficiency in one of the plasma
protein, Tissue factors and calcium interact on the surface coagulation proteins such as factors
of the platelets to form a fibrin clot. VIII.
→Fibrin clot forms at the hemostatic plug to stop the 3 TYPES OF HEMOPHILIA
bleeding. →Lacks FVII
→Platelets provide a surface for the coagulation reaction Hemophilia Type A
(Antihemophilic Factor)
and interact with fibrin to form a stable platelet fibrin clot. →Lacks FIX (Christmas
Type B
Factor)
Factor XIII →Lacks FXI (Plasma
→Coagulation factor that stabilizes the fibrin clot. Type C
Thromboplastin Antecedent)
→Acquired conditions such as Disseminated Intravascular
RESULT OF UNBALANCED HEMOSTATIC SYSTEM Coagulation (DIC), liver and kidney diseases.
→There is thrombus formation →Most of the coagulation factors are synthesized in the
Clotting
(thrombosis) that can occlude liver.
Bleeding →Hemorrhage →Hypocoagulation: Poor clot formation or inability to form
clot.
! NOTE ! →Excessive Bleeding/Fibrinolysis = Bleeding or
*The major role of the hemostatic system is to maintain a Hemorrhage
complete balance of the body’s tendency toward clotting →Platelet number
and bleeding
*Calcium activates coagulation factors and its absence can CONCEPT OF HYPERCOAGULATION
cause inability to form clot

CONCEPT OF NORMAL COAGULATION

Hypercoagulable State
→Other conditions that are related to uncontrolled
thrombosis
→Lysis is the destruction/dissolution of the →Can be seen in patients who have polycythemia vera
Fibrinolysis
fibrin.
→Formation of clot/thrombus or thrombi →This is related to an appropriate formation of thrombi in
Coagulation
formation/ thrombosis. the vascular vessels that occlude normal blood flow.
Platelet →Its major function is coagulation. →Can result to myocardial infarction (MI), stroke, and
→No fibrinolysis = No bleeding = No hemorrhage death
→No coagulation = No thrombosis
→Normal platelet count COMPONENTS OF THROMBI/THROMBUS
! NOTE ! 1 →Platelets
*The formation and dissolution of thrombi is maintained in 2 →WBC
a delicate balance. 3 →RBC

CONCEPT OF HYPOCOAGULATION →Platelet number (Above reference value) =

Uncontrollable Coagulation = Thrombosis

Hypocoagulable →Conditions associated with excessive

States bleeding/fibrinolysis

20
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PRIMARY AND SECONDARY HEMOSTASIS →Platelet →Interaction and adhesion

Aggregation of platelets to one another
TOPIC OUTLINE to form initial plug at injury
1 Hemostasis site.
2 Phases of Hemostasis
Basic Sequence Events in Primary and Secondary
3 3
Hemostasis after Vessel Injury
4 Primary Hemostasis

HEMOSTASIS ! NOTE !
→Physiological process that involves the stoppage of *Initial plug is the main goal in primary hemostasis.
bleeding. *Steps 1-3 are part of primary hemostasis.
→Fibrin-Platelet →Coagulation factors
2 PHASES OF HEMOSTASIS Plug Formation interact on platelet surface
4 to produce fibrin.
PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS →Fibrin-platelet plug then
→Desquamation and small →Large injuries to blood forms at site of vessel injury
injuries to blood vessels vessels and surrounding →Fibrin →Fibrin clot must be
tissues Stabilization stabilized by coagulation
→Involves vascular intima →Involves platelets and factor XIII
and platelets coagulation system
→Rapid, short-lived →Delayed, long-term
response response
→Damaged or activated →Tissue factor is exposed
endothelial cells on cell membranes 5

BASIC SEQUENCE EVENTS IN PRIMARY AND SECONDARY →Fibrin and Coagulation Factor XIII helps stabilize
HEMOSTASIS AFTER VESSEL INJURY the initial platelet plug to produce fibrin meshwork
! NOTE !
STE EVENT COMMENT *Steps 4 and 5 are part of secondary hemostasis
P that involves the coagulation factors to form fibrin
→Controlled by vessel
smooth muscle PRIMARY HEMOSTASIS
→Vasoconstriction
→Enhanced by chemicals (1) Overview of Primary Hemostasis
secreted by platelets (2) 2 Responses
(a) Vascular Response
(b) Platelet Response
1 (3) Normal Blood Flow in Intact Vessels
(4) Primary Hemostasis
(5) Platelet Secretion/Release Mechanism
(6) Platelet Aggregation

OVERVIEW OF PRIMARY HEMOSTASIS

→Adhesion to exposed
→Platelet Adhesion subendothelial connective
tissue
→Role of blood vessel and platelets in response to vascular
injury.
→Formation of platelet plug.
2 RESPONSES
→This involves the vessel spasm and
vasoconstriction

Substance Released
→Inhibits platelet aggregation
2 Prostacycli →Induces vasodilation
n →Inactivates the process of
Vascular
platelet aggregation
Response
→GP Ib/vWF Adenosine →Induces vasodilation
→Platelet Adhesion: Sticking of platelet to →Inactivates thrombin
nonplatelet →Enhances anticoagulant
→vWF is being released by the ECs one the skin is Thrombo- activity of protein C
damaged that will promote adhesion and the modulin →Important in vascular
release of receptor GP Ib to bind the ECs. spasm and vasoconstriction

21
1

Protein C Endothelial Cells

→Inactivates I and VII
→If it passed through
thrombin, it will be converted
into activated protein C (APC)
Heparan →Enhances activity of
Sulphate antithrombin III
Smooth Muscle Nociceptor
→Converts plasminogen to Collagen I and II
plasmin Nociceptor Receptor
→Responsible for the →Pain receptor that is responsible for the contraction of
Tissue
activation of plasminogen to the smooth muscle that will lead to vasoconstriction.
Plasminoge
plasmin
n Activator
(tPA) 3 Mechanism on How to Prevent Blood from Clotting
Plasmin Nitric Oxide + Prostacyclin
→Responsible for the →ECs release nitric oxide (NO) and prostacyclin
dissolution of fibrin (PG12) that inhibits/inactivates platelets
→Required for platelet 1
adhesion ! NOTE !
→Carrier of Factor VIII *NO + Prostacylin = Inactivated Platelets
Von *Inactivated platelets = It cannot bind with ECs
Platelet Adhesion Heparan Sulfate + Antithrombin III
Willebrand
→Requires the receptor and →ECs release heparan sulfate (HS) which is a
Factor
vWF, GP Ib natural anticoagulant that will bind with
antithrombin III (AT III).
! NOTE ! →Binding of HS and AT III inactivates/degrades
* F VIII :  vWF 2
coagulation Factors II (Prothrombin), IX
13-HODE →Inhibits platelet adhesion (Christmas Factor), and X (Stuart-Power Factor)
→Involves the platelet adhesion and
aggregation ! NOTE !
*HS + AT III = Inactivated Factor II, IX and X
Platelet Adhesion Thrombomodulin-Thrombin = Protein C →
→Exposure of Platelets: Activated Protein C (APC)s
o Collagen →ECs secrete thrombomodulin that binds with the
o Fibronectin protein thrombin (F IIa).
o Basement membrane →Protein C passes through thrombin, hence
converting it into activated protein C (APC)
Collagen Type I and II 3
Platelet
→Synthesized by smooth muscle Activated Protein C (APC)
Response
→Location: Deep regions of BV wall →Inactivates/Degrades Factor V and VIII
→Promotes platelet adhesion, aggregation
and release reaction ! NOTE !
*Thrombomodulin + Thrombin = APC
Release Reaction *APC = Inactivated Factor V and VIII
→Activation of platelets wherein granules
will be released from the platelets. PRIMARY HEMOSTASIS
→The content of the centromere will be
→Refers to the role of blood vessels and platelets in the
released during platelet activation or release
primary formation of platelet plug in response to vascular
mechanism to call more platelets
injury.
NORMAL BLOOD FLOW IN INTACT VESSELS
! NOTE !
*Culprit is vessel injury
STEP EVENT COMMENT
→Controlled by vessel
smooth muscle
1 →Vasoconstriction
→Enhanced by chemicals
secreted by platelets

22
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→Vessel Injury → Vessel Spasm/Constriction →

Vasoconstriction = Slowing down of blood flow.

→Breakage of endothelial cells will lead to the

release of vWF.
→Platelets with the help of von Willebrand Factor
(vWF) and GP Ib (vWF:GP Ib complex) will bind to
the receptor promoting platelet adhesion.

3 Mechanisms in Vessel Injury to Prevent Blood Bernard-Soulier Syndrome

Flow →Condition that has a problem with GP Ib that
Endothelin leads to problem with platelet adhesion.
→Damaged ECs will release endothelin will →Interaction and adhesion
bind to the nociceptors (skin receptors) in →Platelet of platelets to one another to
the smooth muscle cells that will result to Aggregation form initial platelet plug at
cellular mechanism and will then lead to injury site.
contraction then vasoconstriction.
1
! NOTE !
*Endothelin + Nociceptor in SM = Cellular
Mechanism → Contraction →
Vasoconstriction = Prevention of Blood
Flow
Myogenic Action (MA) →Requires GPIIb/IIIa and fibrinogen to promote
→Once it has a direct contact with the platelet aggregation.
3
vessel wall or smooth muscle, it will lead to →After platelet aggregation, platelets will then
contraction. secretes its contents that results to reinforcement
2 of more platelets at the site of injury.
! NOTE !
*Myogenic Action + Direct Contact with SM
= Contraction → Vasoconstriction =
Prevention of Blood Flow
Inflammatory Chemicals (Ex. Histamine)
→Inflammatory chemicals will stimulate
nociceptors that will lead to stimulation of
smooth muscles and result to
Formation of Initial Platelet Plug
vasoconstriction.
→End product of primary hemostasis
3
! NOTE ! PLATELET SECRETION/RELEASE MECHANISM
*Inflammatory Chemical + Nociceptor = →Platelets undergo shape changes (due to activation) with
Stimulated SM → Contraction → the intrusion of numerous pseudopods due to the
Vasoconstriction = Prevention of Blood contraction of microtubules.
Flow →Platelet granules move to the center of the platelet and
→Once there is vessel injury, there will also be fuse to the open canalicular system connected to the outer
inflammation. portion of the platelets.
→Adhesion to exposed
→Platelet Open Canalicular System
2 subendothelial connective
Adhesion →Part of the membranous system together with dense
tissue
tubular system (DTS)
→This is where the content of the platelet will pass
through

23
1

→In this way, the contents of the granules (ADP, serotonin,

β-thromboglobulin, PF4, vWF, PDGF) are released outside.

PLATELET AGGREGATION ADP

→This will lead to the release of Adenosine Diphosphate →Very important for platelet aggregation
(ADP), Thromboxane A2 (TXA2), serotonin (for
contraction) to call more platelets at the site of injury. ! NOTE !
ADP and TXA2 *Phospholipids especially phosphatidylserine are
→These works together to stimulate platelets to go at negatively charged
the site of injury →Phospholipases in the platelet membrane will be
TXA2 and Serotonin activated by collagen and epinephrine
→These will go to the smooth muscle to promote
contraction then vasoconstriction to enhance vascular 1
! NOTE !
spasm. *Collagen and Epinephrine = Activated
Phospholipases
Platelet Reaction →Phospholipids on the platelet membrane will
→Release of platelet granules: hydrolyze to release more arachidonic acid.
o Alpha Granules
o Dense Granules 2
! NOTE !
→Stimulated by: *Hydrolization of phospholipase = Release of
o Collagen I and II arachidonic acid
o Thrombin: Stimulates fibrinogen and F XIII →Arachidonic acid can stimulate platelet
o Epinephrine aggregation that will lead to the conversion of
o Thromboxane A2: For platelet aggregation prostaglandin (PGD).
PLATELET AGGREGATION →Cyclooxygenase from the platelet will metabolize
Release of ADP arachidonic acid to produce prostaglandin and
3
→Release of dense granules in response to collagen, endoperoxides
epinephrine, thrombin and TXA2
→Mediates ADP induced aggregation: GP IIb/IIIa with ! NOTE !
calcium and fibrinogen *Arachidonic Acid + Cyclooxygenase = PGD and
o Calcium: Important Cofactor Endoperoxidases
→PGD and endoperoxidases will be converted to
Glanzmann’s Thrombosthenia Thromboxane A2 which is for platelet aggregation
→Result of GP IIb/IIIa deficiency that leads to problems 4
with platelet aggregation ! NOTE !
*PGD + Endoperoxidases = TXA2
→Fibrinogen binding sites on the platelet
membrane will be exposed and the platelet will
then be stimulated by Ca2+ and Mg
→This will then result to the formation of
hemostatic plug or mesh work
5
Calcium (Cofactor) and Magnesium
→Important for platelet aggregation

! NOTE !
Platelet Aggregation *Fibrin Binding Sites + Ca2+ and Mg = Hemostatic
→Platelet stimulating agents (collagen, ADP, epinephrine, Plug/ Meshwork
thrombin) binds to platelets causing them to adhere to one
another.
SUMMARY OF HEMOSTASIS

24
1

→Upper portion is the primary hemostasis while the lower

portion is the secondary hemostasis
SECONDARY HEMOSTASIS
→Involves the enzymatic activation of series of plasma
proteins in the coagulation system to form a fibrin meshwork.
STEP EVENT COMMENT
→Coagulation factors interact on
→Fibrin-
platelet surface to produce fibrin
4 Platelet Plug
→Fibrin-platelet plug then forms at
Formation
site of vessel injury.
→Fibrin →Fibrin clot must be stabilized by
5
Stabilization coagulation factor XIII

→Fibrin mesh is full of phosphatidylserine that is negatively

SECONDARY HEMOSTASIS charged that will initiate the release of factor XII and will
(1) Coagulation result to the start of the coagulation cascade
(2) Overview of Blood Coagulation COAGULATION FACTORS
(3) Secondary Hemostasis FACTOR NAME
(4) Coagulation Factors I →Fibrinogen
(5) Pathways of Coagulation II →Prothrombin
(a) Intrinsic Pathway III →Tissue Thromboplastin
(b) Extrinsic Pathway IV →Calcium Ions
(c) Common Pathway
V →Labile Factor; Proaccelerin
(6) Maintenance of Blood in Liquid State Inside Blood Vessels
VII →Stable Factor; Proconvertin
(7) Thrombin-Mediated Reactions in Hemostasis
VIII →Antihemophilic Factor
(8) Inhibitors of Coagulation (Anticoagulants)
IX →Christmas Factor
COAGULATION X →Stuart-Power Factor
→It is a process whereby, on vessel injury, plasma proteins, XI →Plasma Thromboplastin Antecedent
tissue factors, and calcium interact on the surface of platelets XII →Hageman Factor
to form a stable platelet-fibrin clot. XIII →Fibrin Stabilizing Factor
→Factor III (Thromboplastin) will be released ! NOTE !
→Platelets also interact with fibrin to form a stable platelet- *Foolish People Try Climbing Long Slopes After Christmas
fibrin clot. Some People Have Fallen
→This is a mechanism consisting of a series of cascading *Factor VI is almost the same with factor V and is therefore
reactions involving development of enzymes from their omitted
precursors (zymogen: inactivated) which will further be *Factor X is where common pathway starts
converted to an activated state (serine proteases: activated). *Factor XII deficiency does not result to bleeding
OVERVIEW OF BLOOD COAGULATION Prekallikerin

High-Molecular-Weight Kininogen (HMWK)

Preferred
Roman Synonyms Biochemistry
Descriptive
I →Fibrinogen →Glycoprotein
II →Prothrombin →Glycoprotein

25
1

→Tissue →Lipoprotein o Xa
→Tissue
III Thrombo- o Xia
Factor
plastin →Transaminase
IV →Calcium →Metal Ions →Factor XIIIa
→Labile →Glycoprotein →Prekallikrein
V →Proaccelerin
Factor
VI →Omitted CLASSIFICATION OF COAGULATION FACTORS BY
→Stable →Glycoprotein PHYSICAL PROPERTIES
factor serum →XII
VII →Proconvertin prothrombin →XI
Contact Group
conversion →Prekallikrein
accelerator →HMWK
→Antihemop →Glycoprotein →II
→Anti- hilic Globulin →VII
VIII hemophilic (AHG) →IX
Prothrombin Group/
Factor (AHF) →Antihemop →X
Vitamin K
hilic Factor A
Dependent Group
→Plasma →Anti- →Glycoprotein ! NOTE !
Thrombo- hemophilic *To remember: 1972
IX plastin Factor B *Together with Protein C and S
Component →Christmas →I
(PTC) Factor →V
Fibrinogen Group
→Power →Glycoprotein →VIII
→Stuart- Factor →XIII
X
Power Factor →Stuart
Factor Group Contact Prothrombin Fibrinogen
→Plasma →Anti- →Beta or XI, XII, II, VII, IX, X I, V, VIII. XIII
Thrombo- hemophilic Gamma Globulin Factors PK,
XI plastin Factor C HMWK
Antecedent Absorbed by
(PTA) AL3OH / NO YES NO
→Glass →Sialo- BaSo4
→Hageman Factor glycoprotein Vit K
XII NO YES NO
Factor (HF) →Contact Dependent
Factor Consumed in
→Laki- →Beta or NO NO (II) YES
→Fibrin Clotting
Lorand Gamma Globulin Found in
XIII Stabilizing
Factor (LLF) Serum or BOTH BOTH PLASMA
Factor (FSF)
→Fibrinase Plasma
→Fletcher →Plasma
→Prekallikerin Factor Protein Contact Groups
→Fitzgerald →Plasma →Cannot be absorbed by aluminum hydroxide (AL3OH)
→High Factor Protein →Found both in serum and plasma
Molecular →Williams →Not consumed in coagulation/clotting
Weight Factor
Kininogen →Flaujeac Prothrombin (1972) Group
Factor →Absorbed by aluminum hydroxide and barium sulfate
CATEGORIES OF COAGULATION FACTORS →Most are consumed during coagulation during
coagulation/clotting except Factor II
CLASSIFICATION OF COAGULATION FACTORS BY o Factor II: 80% is used and 20% becomes residual
HEMOSTATIC FUNCTION prothrombin
Substrate COAGULATION SYSTEM
→This will act upon the enzyme →Each is an enzyme precursor which is usually
designated by a Roman Numeral but also given
Cofactors a name
Inactive
→Helps in activation of coagulation factors Form
Substrate →Fibrinogen (Factor I) ! NOTE !
→Factor V (Labile Factor) *Numbers correspond to order of discovery not
Cofactors order in cascade
→Factor VIII:C (AHF)
→Serine Protease: →Usually designated by the letter “a” after the
Enzymes o Ia Active Roman numeral and may also have a different
o IXa Forms name
o Ex: Ia: Fibrinogen

26
1

→Needed for many reactions in the cascade

o Ex. Calcium, platelet factor 3 (PF3), →Activated by XIIa
Cofactors phospholipids →XIa reacts with calcium (cofactor) to
→Each molecule must be present in sufficient activate factor IX
quantity as well as functioning normally. Factor X: Stuart-Power
Factor XIII: Fibrin Stabilizing Factor

Factor XIII
→This protein stabilizes the linkage between
the fibrin monomers of the blood clot.
→It is present in the plasma, platelets and
apparently synthesized by megakaryocytes.
→Stabilizes the fibrin clot
→Half-Life: 150 hours
→Seen in the common pathway
Factor XII: Hageman Factor
Factor X →Serine protease not consumed in the
clotting process found in both serum and
plasma.
→Pptd by 55%-65% ammonium sulfate.
→Activated by the products of both extrinsic
and intrinsic
→Half-Life:48-52 hrs

→Activated by either intrinsic or extrinsic

→Where the common pathway starts
Factor IX: Christmas Factor

→A stable globulin that is not consumed

→This factor is activated by contact with
Factor XII foreign surfaces and initiates the intrinsic Factor IX
system.
→It is also involved in the activation of
fibrinolysis.
→Serine protease →Present in both serum or plasma
→Half-Life: 48-70 hrs →Vit K dependent
→Not consumed during clotting
→Contact factors (prekallikrein and HMWK) →Essential component of the intrinsic
→Prekallikrein will be activated by HMWK thromboplastin generating system
into kallikrein that will activate factor XII →Half-Life: 24 hrs
→Factor XIIa will loop back to the contact Factor VIII: Antihemophilic Factor
factors to produce more activated Factor XII
→This is the first coagulation factor to appear
after platelet aggregation to initiate the
coagulation cascade.
Factor XI: Plasma Thromboplastin Antecedent

Factor VIII

→Name: Anti-hemophilic factor/

antihemophilic factor A
Factor XI
→Source: Endothelium lining blood vessel
and platelet
→Activator: Thrombin
→Actions: Works with Factor IX and calcium
→Serine protease beta or gamma globulin to activate Factor X
partly consumed in clotting found in serum Factor VII Factor VII: Stable Factor
and plasma
→Half-Life: 48-84 hrs

27
1

→Stable factor
→It is activated by tissue thromboplastin
which activates factor X →Proenzyme, precursor of thrombin
→Vit K dependent →Vit k is important for the production of
→Half-Life: 6 hrs prothrombin by the liver.
Vit K dependent
→Part of extrinsic pathway →Half-Life 60 hrs
Factor V: Labile Factor →MPC: 10-20 mg/dl.
Factor I: Fibrinogen

→Synthesized in the liver

→Unstable when stored in citrated plasma.
Factor V →Consumed during clotting process not
found in the serum. Factor I
→Half-Life: 24 hours
→The substrate for thrombin and precursor
→MPC: 1mg/dl
of fibrin.
→Function is to be converted into a soluble
→Sample must be run ASAP to prevent
protein
activation or loss of coagulation factors
→Absent in serum 75%
→Tests: PT and APTT
→Present in the plasma
→Combines with calcium and phospholipid to
→Half-Life: 100-150 hrs
activate prothrombin
→MPC: 200-400 mg/dl
Factor IV: Calcium Ions
→Contact factor
→It is an active form of calcium that is
→This is a plasma protein with a molecular
Factor IV required for the activation of thromboplastin
weight of approximately 85,000 Daltons.
and for conversion of prothrombin
→It is partially adsorbed by barium sulfate
→MPC: 8-l0 mg/dl Prekallikrein
but not adsorbed by aluminum hydroxide.
Factor III: Tissue Thromboplastin
→Activator of factor xi in conjunction with w/
HMWK
→Half-Life: 35 hrs
High →Contact factor
Molecular →Fitzgerald Factor
Weight →Activator of factor xi in effect w/
Kininogen prekallikerin
Factor III (HMWK)
PATHWAYS OF COAGULATION

→Tissue factor cofactor half-life insoluble

used to designate the clot accelerating action
of extract of tissue.
→Thromboplastin is required for the
Extrinsic
conversion of prothrombin to thrombin
Pathway
Factor II Factor II: Prothrombin

→Activated by the release of Factor III (TF,

Tissue Thromboplastin) into the plasma from
injured tissue
→Takes place for 30 seconds
Intrinsic
Pathway

28
1

→Xi
→IX
→VIII
→X
→III
Extrinsic →VII
→X
→X
→V
Common
→II
→I
→Activation occurs when a vessel is injured,
MAINTENANCE OF BLOOD IN LIQUID STATE INSIDE BLOOD
exposing the subendothelial basement membrane
and collagen. VESSELS
→This will lead to the activation of the “Contact →Thrombin formation marks a critical event in hemostatic
Factor”, Factor XII together with Factor XII, process.
HMWK and prekallikrein
→Takes place for 4-6 mins Actions of Thrombin
→Converts fibrinogen to fibrin
→Activated Factor XIII
→Enhances the activity of Factors V and VIII:C
→Induces platelet aggregation
THROMBIN-MEDIATED REACTIONS IN HEMOSTASIS
→Induces platelet activation and
aggregation.
Common
→Activates cofactor VIII to VIIIa
Pathway Procoagulant
→Converts Factor XIII to XIIIa
→Via autocatalysis converts Prothrombin to
→This pathway starts with the activation of Thrombin
Factor X to Factor Xa by either intrinsic or
→Binds to antithrombin to inhibit serine
extrinsic pathway.
Coagulation proteases
Inhibitor →Binds to thrombomodulin to activate
PL: Phospholipids
protein C (inhibits Va and VIIIa)
→Induces cellular chemotaxis
→Stimulates proliferation of smooth muscle
and endothelial cells.

Chemotaxis
Positive →Cell goes toward the
Tissue Repair Chemotaxis site of injury
Negative →Cell goes away the site
Chemotaxis of injury

Retraction and Fibrinolysis

→Leads to proliferation of smooth muscle
and endothelial cells.
INHIBITORS OF COAGULATION (ANTICOAGULANTS):
PHYSIOLOGIC INHIBITORS
→Glycoprotein that is produced by the liver
and is a major inhibitor of blood
coagulation.
Protein C
→This inactivates factors VIII:C and Va in
the presence of cofactor Protein S.
→Vitamin K dependent
→A vitamin K dependent protein and also
produced by the liver.
→This functions to enhance binding of
Protein S
Protein C to phospholipid surfaces and
increase the rate of Factors Va and VIIIa
inactivation by Protein C.
Factors Involved in Coagulation Pathway Thrombo- →Inhibits thrombin and inactivates the
Intrinsic →XII modulin clotting cascade.

29
1

Antithrombin →Major inhibitor of Thrombin and Factor

III Xa
Heparin →Inhibits Thrombin
Cofactor →Activity is enhanced by heparin
Alpha 2 →Forms complex with thrombin, kallikrein,
Macroglobulin thus inhibiting their activities.
Extrinsic →Lipoprotein associated inhibitor (LACI)
Pathway that inhibits VIIa which is a tissue complex
Inhibitor factor
→Inactivates Factor XIIa and plasma
kallikrein.
→Factor XIa and plasmin
C1 Inhibitor
Plasmin
→Activated form of plasminogen
Alpha1 →A slow reacting thrombin inhibitor which
Antitrypsin inhibits Factor XIa and Xa
Activated →Inhibits Protein C.
Protein C →This is more active with heparin present.
Inhibitor

! NOTE !
*Inhibitors and activators are important to maintain balance
and prevent thrombosis and hemorrhage.
*Protein C and S works synergistically

30
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FIBRINOLYSIS Plasminogen →Comes from the liver

(Profirbino-
TOPIC OUTLINE lysin)
1 Fibrinolysis Plasmin →Proteolytic enzymes
2 Primary Fibrinolysis (Fibrinolysin)
3 Secondary Fibrinolysis →Neutralize the activity of plasmin
4 Components of Fibrinolytic System →The major inhibitor
5 The Actions of Plasmin of free plasmin.
6 Concept of Fibrinolysis →It is present in the
7 Degradation of Fibrin and Noncross-Linked Fibrin plasma and also in
Alpha2
platelets.
Antiplasmin
FIBRINOLYSIS →Synthesized in the
→It is a system whereby the temporary fibrin clot is liver
systematically and gradually dissolved as the vessel heals in →Serine protease
order to restore normal blood flow. inhibitor
→Last and final stage of coagulation. →Inactivates the
→Lysis will lead to destruction of fibrin to go back to normal Alpha2 plasmin that is not
blood flow. Macroglobulin inhibited by alpha2
→The star is plasmin. antiplasmin
Inhibitors of
→Released by
Fibrinolysis
2 Major Proteins Important for Lysis platelets, it inhibits
Thrombospondin
→Tissue Plasminogen Activator (TPA) activation of fibrin-
→Urokinase Plasminogen Activator (UPA) bound plasminogen.
→Both are naturally
Plasmin occurring
Plasminogen
→This will help remove/lyse the fibrin. →They come from
Activator
endothelial cells and
Inhibitor 1 (PAI-
PRIMARY FIBRINOLYSIS platelets.
1) and
→Excessive amounts of plasminogen activators from →Inactivates TPA and
Plasminogen
damaged cells/malignant cells UPA
Activator
→Converts plasminogen to plasmin in the absence of fibrin →Control or prevent
Inhibitor 2 (PAI-
formation. plasminogen to be
2)
→Also known as normal fibrinolysis. converted into
→Plasminogen will be converted into plasmin (active form) plasmin

SECONDARY FIBRINOLYSIS THE ACTIONS OF PLASMIN

Disseminated Intravascular Coagulation (DIC)
→Uncontrolled, inappropriate formation of fibrin within the →Destroys fibrinogen and fibrin
1
blood vessels →Degradation of fibrinogen and fibrin
→Seen in patients with cancer and is the secondary condition →Produces Fibrin Degradation Products (FDP), which
→Continuous bleeding 2 increase vascular permeability and interfere with
Conditions Associated thrombin-induced fibrin formation.
→Infection →Produces D-Dimer
→Neoplasm
→Snake bite D-Dimer
→HTR 3
→Consists of 2 D domains
→Produced from the cross-link made by F XIII
COMPONENTS OF FIBRINOLYTIC SYSTEM →Used to determine if the patient has clot
→There should be a control of fibrinolysis to prevent bleeding →Destroys Factor V, VIII, IX, XI, and other plasma
and hemorrhage. 4
proteins
→Half-Life: 24-26 hrs →Indirectly enhances or amplifies conversion of
→PAI-1 and -2 5
Factor XII to XIIa
→Tissue plasminogen →Enhances or amplifies conversion of PK to
activator (TPA) 6
kallikrein, liberating from kininogen
→Single-chain urokinase- 7 →Cleaves C3 fragments.
Endogenous
Plasminogen like plasminogen activator
Activators →Two-chain urokinase CONCEPT OF FIBRINOLYSIS

→Streptokinase
→Acy-Plasminogens
Exogenous
Streptokinase Activator
Complex (APSAC)

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→The earliest proteolytic activity results in still-clottable

X-fragment, which is subsequently degraded to the
unclottable Y & D fragments

Y Fragment
→Consisting of D plus E portions, is the itself split into
these components.

→Small peptides are also produced at a number of the

proteolytic cleavage.
Factor XII CONCEPT OF DEGRADATION OF FIBRIN AND NONCROSS-
→Reacted with collagen contact to be activated (XIIa) LINKED FIBRIN (NOT STABILIZED BY F XIII)
1
→Also activated and producedwith the help of other
contact factors (prekallikrein and HMWK)
Activation of Plasminogen
2 →Kallikrein and tissue activation will activate
plasminogen to plasmin
Degradation of Fibrinogen
3
→Plasmin degrades fibrinogen to fibrin
Fibrin Degradation Products
4 →Degradation of fibrinogen produce fibrin split
degradation products.

Fibrinogen/Fibrin (Urea Soluble)

→Degraded by plasmin to form fragment X

Fragment X
→Degraded by plasmin to form fragment Y and D

Fragment Y
→Degraded by plasmin to form fragment D and E

Activation of Plasminogen: Plasminogen Actovators

→tPA, PAI-1 and 2, urokinase (single-chain), Factor
1
XIa, XIIa, and kallikrein converts plasminogen to
plasmin
Control of Fibrinolysis: Inhibitors of Fibrinolysis
→α2-Antiplasmin and α2-Macroglobulin act upon
plasmin to prevent its overactivation
2
Free Plasmin
→Can also digest fibrinogen
Degradation of Fibrin
→Thrombin activates fibrinogen
→Thrombin-activatable fibrinolysis inhibitor will act
3
upon fibrin to help in its degradation.
→Fibrin degradation products will then be the end
product.
→When a normally cross-lined fibrin clot is dissolved by
plasmin, isolated D and E fragments are not the
characteristic end stage fragments.
→Rather, a variety of complexes are found, most
characteristically one composed of two D and one E
moieties (D2E Fragment), which is called a D-dimer.
D-Dimer
→Assay is done to determine if all of the clots are degraded
! NOTE !
*Finale Product: D-dimer
*Complex should be in order (DD/E not E/DD)

DEGRADATION OF FIBRIN AND NONCROSS-LINKED FIBRIN

DEGRADATION OF FIBRIN AND NONCROSS-LINKED

FIBRIN
→Plasmin degrades fibrin clot during fibrinolysis and also
native fibrinogen in a process called fibrinogenolysis.

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PRINCIPLE OF COAGULATION; PRIMARY AND SECONDARY →These proteins circulate as inactive

HEMOSTASIS; AND FIBRINOLYRIS zymogens (proenzymes) that become
activated during the process of coagulation
Book Based and, in turn, form complexes that activate
other zymogens to ultimately generate
TOPIC OUTLINE thrombin
1 Overview of Hemostasis
2 Vascular Intima in Hemostasis Thrombin
3 Platelets →An enzyme that converts fibrinogen to a
4 Coagulation System localized fibrin clot.
5 Coagulation Regulatory Mechanisms
6 Fibrinolysis Fibrinolysis
→The final event of hemostasis
OVERVIEW OF HEMOSTASIS →The gradual digestion and removal of the
fibrin clot as healing occurs.
HEMOSTASIS PRIMARY SECONDARY
→It is a complex physiologic process that keeps circulating →Activated by →Activated by large
blood in a fluid state and then, when an injury occurs, desquamation and small injuries to blood vessels
produces clot to stop the bleeding, confines the clot to the injuries to blood vessels. and surrounding tissues.
site of injury, and finally dissolves the clot as the wound →Involves vascular intima →Involves platelets and
heals. and platelets coagulation system.
→When hemostasis is out of balance, hemorrhage →Rapid, short-lived →Delays, long-term
(bleeding) or thrombosis (pathological clotting) can be life- response response
threatening. →Procoagulant substances →The activator, tissue
→Absence of a single procoagulant may lead to lifelong: exposed or released by factor, is exposed on cell
o Anatomic Hemorrhage damaged or activated by membranes
o Chronic Inflammation endothelial cells
o Transfusion Dependence
→Absence of control protein allows coagulation that may VASCULAR INTIMA IN HEMOSTASIS
lead to: (1) Vascular Intima
o Thrombosis (2) Anticoagulant Properties of Intact Vascular Intima
o Stroke (3) Procoagulant Properties of Damaged Vascular Intima
o Pulmonary Embolism (4) Fibrinolytic Properties of Vascular Intima
o Deep Vein Thrombosis
o Cardiovascular Events (such as myocardial VASCULAR INTIMA
infarction) →It provides the interface between circulating blood and
→Plasma components are the coagulation and fibrinolytic the body tissues.
proteins and their inhibitors. →The innermost lining of blood vessels is a monolayer of
PRIMARY AND SECONDARY HEMOSTASIS metabolically active endothelial cells (EC).
→The role of blood vessels and platelets in
response to a vascular injury, or to the Endothelial Cells
common place desquamation of dying or →Complex and heterogeneous and are distributed
damaged endothelial cells. throughout the body.
→Blood vessels contract to seal the wound →They display unique structural and functional
or reduce the blood flow (vasoconstriction). characteristics, depending on their environment and
→Platelets become activated, adhere to the physiologic requirements, not only in subsets of blood
site of injury, secrete the contents of their vessels such as arteries versus veins but also in the various
granules, tissues and organs of the body.
and aggregate with other platelets to form a →ECs form a smooth, unbroken surface that eases the fluid
Primary
platelet plug. passage of blood.
Hemostasis
→Vasoconstriction and platelet plug →An elastin-rich internal elastic lamina (basement
formation comprise the initial, rapid, short- membrane) and its surrounding layer of connective tissues
lived response to vessel damage, but to support the ECs.
control major bleeding in the long term, the →In all blood vessels, fibroblasts occupy the connective
plug must be reinforced by fibrin. tissue layer and produce collagen.
→Defects in primary hemostasis such as →Smooth muscle cells in arteries and arterioles, but not in
collagen abnormalities, thrombocytopenia, the walls of veins, venules, or capillaries, contract during
qualitative platelet disorders, or von primary hemostasis.
Willebrand disease can cause debilitating, ANTICOAGULANT PROPERTIES OF INTACT VASCULAR
sometimes fatal, chronic hemorrhage. INTIMA
→The activation of a series of coagulation →Normally, the intact vascular endothelium prevents
Secondary
proteins in the plasma, mostly serine thrombosis by:
Hemostasis
proteases, to form a fibrin clot. o Inhibiting platelet aggregation

33
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o Preventing coagulation activation and undesirable platelet activation in intact

propagation vessels.
o Enhancing fibrinolysis →Synthesized in ECs, vascular smooth
→Several specific anticoagulant mechanisms prevent muscle cells, neutrophils, and macrophages.
intravascular thrombosis. →It induces smooth muscle relaxation and
Nitric Oxide
subsequent vasodilation, inhibits platelet
ANTICOAGULANT PROPERTIES OF INTACT activation, and promotes angiogenesis and
ENDOTHELIUM healthy arterioles.
→Composed of rhomboid cells presenting a Tissue →An important EC-produced anticoagulant.
1
smooth, contiguous surface Factor →It controls activation of the tissue factor
→Secretes the eicosanoid platelet inhibitor Pathway pathway
2
prostacyclin Inhibitor
3 →Secretes vascular “relaxing” factor nitric oxide (TFPI)/
→Secretes the anticoagulant glycosaminoglycan External
4 Coagulative
heparan sulfate
→Secretes coagulation extrinsic pathway regulator Pathway
5 →Synthesize and express on EC’s surfaces
tissue factor pathway inhibitor
6 →Expresses endothelial protein C receptor inhibitors of thrombin formation
→Expresses cell membrane thrombomodulin, a →Facilitated by endothelial protein C
7 receptor (EPCR), and heparan sulfate.
protein C coagulation control system activator
→Binds protein C, and
thrombomodulin catalyzes
the activation
of the protein C pathway.
Endothelia
l Protein C Protein C Pathway
Receptor →Downregulates
coagulation by digesting
activated factors V and VIII,
thereby inhibiting thrombin
formation.
→A glycosaminoglycan that
enhances the activity of
Thrombo-
antithrombin,
modulin
Antithrombin
VASCULAR INTIMA OF THE BLOOD VESSELS →A serine protease inhibitor.
Innermost →Endothelial Cells (Endothelium)
Vascular Heparin
Lining →The pharmaceutical
Supporting the →Internal elastic lamina composed of Heparan anticoagulant manufactured
Endothelial elastin and collagen Sulfate from porcine gut tissues,
Cells resembles EC heparan sulfate
Subendothelial Veins →Collagen and fibroblasts in its antithrombin activity.
Connective →Collagen, fibroblasts, and →It is used extensively as a
Arteries
Tissue smooth muscle cells therapeutic agent to prevent
propagation of the thrombi
Endothelial System that cause coronary
→Rhomboid and contiguous, providing a smooth inner thrombosis, strokes, deep
surface of the blood vessel that prevents harmful vein thromboses, and
turbulence that otherwise may activate platelets and pulmonary emboli.
coagulation enzymes. PROCOAGULANT PROPERTIES OF DAMAGED VASCULAR
→It forms a physical barrier separating procoagulant INTIMA
proteins and platelets in blood from collagen in the internal →Although the intact endothelium has anticoagulant
elastic lamina that promotes platelet adhesion, and tissue properties, when damaged, the vascular intima promotes
factor in fibroblasts and smooth muscle cells that activates coagulation.
coagulation.
SUBSTANCES SYNTHESIZED AND SECRETED BY EC TO → Any harmful local stimulus, whether
MAINTAIN NORMAL BLOOD FLOW mechanical or chemical, induces vasoconstriction
→A platelet inhibitor and a vasodilator, is in arteries and arterioles.
Prostacycli 1
synthesized through the eicosanoid →Smooth muscle cells contract, the vascular
n
pathway and prevents unnecessary or lumen narrows or closes, and blood flow to the
injured site is minimized.

34
1

→Although veins and capillaries do not have Exposed smooth →Tissue factor exposed on cell
smooth muscle cells, bleeding into surrounding muscle cells and membranes
tissues creates extravascular pressure on the fibroblasts
blood vessel, effectively minimizing the escape of ECs in →Tissue factor is induced by
blood. inflammation inflammation
→The subendothelial connective tissues of
arteries and veins are rich in collagen, a flexible, FIBRINOLYTIC PROPERTIES OF VASCULAR INTIMA
elastic structural protein that binds and activates →ECs support fibrinolysis, the removal of fibrin to restore
2 platelets. vessel patency, with the secretion of tissue plasminogen
→Some connective tissue degeneration occurs activator (TPA).
naturally in aging, which leads to an increased →During thrombus formation, both TPA and plasminogen
bruising tendency. bind to polymerized fibrin.
→ECs secrete von Willebrand factor (VWF) from
storage sites called Weibel-Palade bodies when Tissue Plasminogen Activator (TPA)
activated by vasoactive agents such as thrombin. →It activates fibrinolysis by converting plasminogen to
3 plasmin, which gradually digests fibrin and restores blood
VWF flow.
→A large multimeric glycoprotein that is
necessary for platelets to adhere to exposed →ECs also regulate fibrinolysis by providing inhibitors to
subendothelial collagen in arterioles. prevent excessive plasmin generation.
→On activation, ECs secrete and coat themselves →ECs, as well as other cells, secrete plasminogen activator
with P-selectin, inhibitor 1 (PAI-1),

P-selectin Plasminogen Activator Inhibitor 1 (PAI-1)

→An adhesion molecule that promotes platelet →A TPA control protein that inhibits plasmin generation
and leukocyte binding. and fibrinolysis.
4
→Secreted by endothelial cells (ECs).
Immunoglobulin-Like Adhesion Molecules
(ICAMs) and Platelet Endothelial Cell Adhesion Thrombin-Activator Fibrinolysis Inhibitor (TAFI)
Molecules (PECAMs) →Another inhibitor of plasmin generation that is activated
→Also secreted by ECs that further promote by thrombin bound to EC membrane thrombomodulin.
platelet and leukocyte binding.
→Finally, subendothelial smooth muscle cells and →Elevations in PAI-1 or TAFI can slow fibrinolysis and
5 fibroblasts support the constitutive membrane increase the tendency for thrombosis.
protein tissue factor. →Although the significance of the vascular intima in
hemostasis is well recognized, there are few valid
→Physiologically, EC disruption exposes tissue factor in laboratory methods to assess the integrity of ECs, smooth
subendothelial cells and activates the coagulation system muscle cells, fibroblasts, and their collagen matrix.
through contact with plasma factor VII. →The diagnosis of blood vessel disorders is often based on
→In pathological conditions, tissue factor may also be the following that rule out platelet or coagulation
expressed on bloodborne monocytes during inflammation disorders:
and sepsis and by tissue factor–positive microparticles o Clinical symptoms
derived from membrane fragments of activated or o Family history
apoptotic vascular cells and possibly on the surface of some o Laboratory tests
ECs.
→Activation of the TF:VIIa:Xa complex within the PLATELETS
circulation is limited by TFPI. (1) Platelets
(2) Platelet Functions
PROCOAGULANT PROPERTIES OF THE DAMAGED (a) Platelet Adhesion
STRUCTURE PROCOAGULANT PROPERTY (b) Platelet Aggregation
Smooth muscle →Induce vasoconstriction (c) Platelet Secretion
cells in arterioles (3) Coagulation
and arteries
Exposed →Binds VWF and platelets →Platelets are produced from the cytoplasm of bone marrow
subendothelial Megakaryocytes.
collagen →Although platelets are only 2 to 3 mm in diameter on a
→Secrete VWF fixed, stained peripheral blood film, they are complex,
→Secrete adhesion molecules: metabolically active cells that interact with their environment
Damaged or and initiate and control hemostasis.
o P-selectin
activated ECs →At the time of an injury, platelets adhere, aggregate, and
o ICAMs
o PECAMs secrete the contents of their granules.

PLATELET ADHESION

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Adhesion
→The property by which platelets bind nonplatelet PLATELET GRANULE CONTENTS
surfaces such as subendothelial collagen. Platelet Dense
Platelet Alpha Granules
Granules/Dense Bodies
→Further, VWF links platelets to collagen in areas of high (Large Molecules)
(Small Molecules)
shear stress such as arteries and arterioles, whereas Adenosine Diphosphate
platelets may bind directly to collagen in damaged veins β-Thromboglobulin (Activates Neighboring
and capillaries. Platelets)
Factor V Adenosine Triphosphate
Von Willebrand Factor (VWF) Factor XI Calcium
→VWF binds platelets through their glycoprotein GP Serotonin
Ib/IX/V membrane receptor. Protein S
(Vasoconstrictor)
Fibrinogen
→The importance of platelet adhesion is underscored by VWF
bleeding disorders such as Bernard-Soulier syndrome and
Platelet Factor 4 (Heparin
von Willebrand disease
Inhibitor)
Platelet-Derived Growth
Bernarn-Soulier Syndrome
Factor
→In which the platelet GP Ib/IX/V receptor is absent
→During activation, ADP and Ca2+ activate phospholipase
von Willebrand Disease
A2,
→In which VWF is missing or defective.
PLATELET AGGREGATION
Phospholipase A2
Aggregation →Converts membrane phospholipid to arachidonic acid.
→The property by which platelets bind to one another.
Cyclooxygenase
→When platelets are activated, a change in the GP IIb/IIIa
→Converts arachidonic acid into prostaglandin
receptor allows binding of fibrinogen, as well as VWF and
endoperoxides.
fibronectin.
→Fibrinogen binds to GP Iib/ IIIa receptors on adjacent
Thromboxane Synthetase
platelets and joins them together in the presence of ionized
→Converts prostaglandins into thromboxane A2 (TX2),
calcium (Ca2+). which causes Ca2+ to be released and promotes platelet
aggregation and vasoconstriction
Fibrinogen Binding
→It is essential for platelet aggregation, as evidenced by Aspirin Acetylation
bleeding and compromised aggregation in patients with: →Permanently inactivates cyclooxygenase, blocking
o Afibrinogenemia
thromboxane A2 production and causing impairment of
o Patients who lack the GP Iib/IIIa receptor
platelet function (aspirin effect).
(Glanzmann thrombasthenia)
COAGULATION
→The platelet membrane is the key surface for coagulation
MOST COMMONLY USED AGONISTS TO INDUCE
enzyme-cofactor-substrate complex formation.
AGGREGATION IN IN VITRO PLATELET AGGREAGTION
→Platelets supply Ca2+, the membrane phospholipid
→Thrombin or Thrombin Receptor Actovation
1 phosphatidylserine, procoagulant factors, and receptors.
Peptide (TRAP)
2 →Arachidonic acid
→Coagulation is initiated on tissue factor–bearing
3 →Adenosine diphosphate (ADP) cells (such as fibroblasts) with the formation of
4 →Collagen the extrinsic tenase complex TF:VIIa:Ca2+, which
5 →Epinephrine 1
activates factors IX and X and produces enough
→These bind to their respective platelet membrane thrombin to activate platelets and factors V, VIII,
receptors. and XI in a feedback loop.
PLATELET SECRETION →Coagulation is then propagated on the surface of
→Platelets secrete the contents of their granules during the platelet with the formation of the intrinsic
adhesion and aggregation, with most secretion occurring tenase complex (Ixa:VIIIa:phospholipid:Ca21) and
late in the platelet activation process. 2
the prothrombinase complex
(Xa:Va:phospholipid:Ca21), ultimately generating
COMPONENTS SECRETED BY PLATELETS a burst of thrombin at the site of injury.
→Factor V
→VWF →Erythrocytes, monocytes, and lymphocytes also
Procoagulants
→Factor VIII participate in hemostasis.
→Factor I (Fibrinogen)
→Ca2+ Erythrocytes
Control
→ADP, →Add bulk and structural integrity to the fibrin clot; there
Proteins
→Other hemostatic molecules is a tendency to bleed in anemia.

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Monocytes, Lymphocytes, and Endothelial Cells (Ecs) Activated Procoagulant

→In inflammatory conditions, these provide surface-borne →When a procoagulant becomes activated, a lowercase a
tissue factor that may trigger coagulation. appears behind the numeral
→Leukocytes also have a series of membrane integrins and o Example:Activated factor VII is VIIa.
selectins that bind adhesion molecules and help stimulate →Both zymogens and cofactors become activated in the
the production of inflammatory cytokines that promote the coagulation process.
wound-healing process.
! NOTE ! Factors I and II
*Platelet Adhesion: Platelet to Other Substances →We customarily call factor I fibrinogen and factor II
*Platelet Aggregation: Platelet to Platelet prothrombin, although occasionally they are identified by
their numerals.
SUMMARY OF PLATELET FUNCTION o Factor I: Fibrinogen
FUNCITON CHARACTERISTICS o Factor II: Prothrombin
→Reversible
Adhesion Factor III: Thromboplastin
→Seals endothelial gaps, some
→Platelets roll and →A crude mixture of tissue factor and phospholipid.
secretion of growth factors, in
cling to nonplatelet
arterioles VWF is necessary for
surfaces →Now that the precise structure of tissue factor has been
adhesion
→Irreversible described, the numeral designation is seldom used.
Aggregation →The numeral IV identified the plasma cation calcium
→Platelet plugs form, platelet
→Platelets adhere to (Ca2+); however, calcium is referred to by its name or
contents are secreted, requires
each other chemical symbol, not by its numeral.
fibrinogen
Secretion →Irreversible
→Platelets discharge →Occurs during aggregation, Removal of Factor VI
the contents of their platelet contents are secreted, →The numeral VI was assigned to a procoagulant that later
granules essential to coagulation was determined to be activated factor V
→VI was withdrawn from the naming system and never
COAGULATION SYSTEM reassigned.
(1) Nomenclature of Procoagulants
(2) Classification and Function of Procoagulants Factor VIII (Antihemophilic Factor)
(a) Vitamin K-Dependent Prothrombin Group →A cofactor that circulates linked to a large carrier
(b) Cofactors in Hemostasis protein,
(c) Factor VII and Von Willebrand Factor
(d) Factor XI and the Contact Factors Other Factors Involved in the Coagulation System
(e) Thrombin →The following have never received Roman numerals
(f) Fibrinogen Structure and Fibrin Formation, Factor XIII because they belong to the kallikrein and kinin systems,
(3) Plasma-Based (In Vitro) Coagulation: Extrinsic, Intrinsic, respectively, and their primary functions lie within these
and Common Pathways systems.
(4) Cell-Based (In Vivo, Physiologic) Coagulation o vWF: Von Willebrand Factor
(a) Initiation o Prekallikrein (Pre-K): Fletcher Factor
(b) Propagation o High-Molecular-Weight Kininogen (HMWK):
Fitzgerald
NOMENCLATURE OF PROCOAGULANTS
Platelet Phospholipids: Platelet Factor 3 (PF3)
→Plasma transports at least 16 procoagulants, also called
→Platelet phospholipids, particularly phosphatidylserine,
coagulation factors.
are required for the coagulation process but were given no
→Nearly all are glycoproteins synthesized in the liver,
although monocytes, ECs, and megakaryocytes produce a Roman numeral; instead they were once called collectively
few. platelet factor 3.
→Eight are enzymes that circulate in an inactive form
called zymogens.
→Others are cofactors that bind, stabilize, and enhance the
activity of their respective enzymes.
→During clotting, the procoagulants become activated and
produce a localized thrombus.
→In addition, there are plasma glycoproteins that act as
controls to regulate the coagulation process.

International Committee for the Standardization of the

Nomenclature of the Blood Clotting Factors (In 1958)
→They officially named the plasma procoagulants using
Roman numerals in the order of their initial description or
discovery.

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TABLE OF PLASMA PROCOAGULANTS

38
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CLASSIFICATION AND FUNCTION OF PROCOAGULANTS →Required for the assembly of coagulation complexes
→The plasma procoagulants may be serine proteases or on platelet or cell membrane phospholipids.
cofactors, except for factor XIII, which is a →Serine proteases bind to negatively charged
transglutaminase. phospholipid surfaces, predominantly phosphatidylserine,
through positively charged calcium ions.
Serine Proteases →Activation is a localized cell-surface process, limited to
→Proteolytic enzymes of the trypsin family and include the the site of injury and controlled by regulatory mechanisms.
procoagulants: →If zymogen activation is uncontrolled and generalized,
o Thrombin (Factor IIa) the condition is called disseminated intravascular
o Factors VIIa, IXa, Xa, XIa, and XIIa coagulation (DIC), a serious, often life-threatening
o Pre-K condition.
→Each member has a reactive seryl amino acid residue in
its active site and acts on its substrate by hydrolyzing →These essential pieces of clinical information assist in the
peptide bonds, digesting the primary backbone, and interpretation of laboratory tests, monitoring of
producing smaller polypeptide fragments. anticoagulant therapy, and design of effective replacement
→Serine proteases are synthesized as inactive zymogens therapies in deficiency-related hemorrhagic diseases.
consisting of a single peptide chain. →For example, factor VIII has a short half-life of 12 hours,
→Activation occurs when the zymogen is cleaved at one or so replacement therapy for hemophilic individuals who are
more specific sites by the action of another protease during deficient in factor VIII is administered every 12 hours.
the coagulation process. →For most factors, the level that achieves hemostatic
effectiveness is 25% to 30%.
PLASMA PROCOAGULANT SERINE PROTEASES →This is the minimum level that must be maintained to
Inactive Active prevent bleeding in factor-deficient patients.
Cofactor Substrate →Therapy for a hemophilic patient is designed to maintain
Zymogen Protease
Fibrinogen, the factor level above 30%.
Prothrombin Thrombin →A higher level may be desirable, such as in a patient
V, VIII, XI,
(II) (IIa) preparing for surgery.
XIII
Tissue
VII VIIa IX, X Half-life of Procoagulant Factors
Factor
IX IXa VIIa X →The half-life is also important in monitoring
X Xa Va Prothrombin anticoagulant therapy, especially warfarin (Coumadin),
because even though factor VII becomes reduced in 6
XI Xia IX hours, the reduction of prothrombin takes 4 to 5 days.
→Therefore, the full effect of warfarin is not realized until
XII XIIa HMWK XI approximately 5 days after therapy has begun.
Prekallikrein Kallikrein HMWK XI VITAMIN K-DEPENDENT PROTHROMBIN GROUP
Prothrombin Group
Tissue Factors
VITAMIN K-DEPENDENT COAGULATION FACTORS:
→The procoagulant cofactors that participate in complex
PROTHROMBIN GROUP
formation are tissue factor, located on membranes of
→Prothrombin (II)
fibroblasts and smooth muscle cells, and soluble plasma
→VII
factors V, VIII, and HMWK. Procoagulants
→IX
→X
TISSUE FACTORS
→Protein C
1 →Tissue Factor Regulatory
→Protein S
2 →Factor V (Labile Factor) Proteins
→Protein Z
3 →Factor VIII (Antihemophilic Factor) →These are named the prothrombin group because of
4 →High-Molecular-Weight Kininogen their structural resemblance to prothrombin.
→All seven proteins have 10 to 12 glutamic acid units near
OTHER PLASMA PROCOAGULANTS their amino termini.
1 →Fibrinogen
2 →Factor XIII (Fibrin Stabilizing Factor/FSF) →Prothrombin (II)
3 →Phospholipids Serine Protease
→VII, IX, X
4 →Calcium When Activated
→Protein C
5 →von Willebrand Factor (vWF) →Protein S
Cofactors
→Protein Z
Fibrinogen
→The ultimate substrate of the coagulation pathway. Vitamin K
→When hydrolyzed by thrombin, fibrinogen forms the →It is a quinone found in green leafy vegetables and is
primary structural protein of the fibrin clot, which is produced by the intestinal organisms Bacteroides fragilis
further stabilized by factor XIII. and Escherichia coli.
→Food Sources High in Vitamin K
Calcium o Kale o Asparagus

39
1

o Spinach o Cabbage →More Efficiently Activated Factor: Factor

o Turnip greens o Green onions X
o Lettuce: Boston, →It is composed of factor Xa and its
o Collards
Romaine, or Bibb cofactor Va
o Mustard greens o Avocado →This converts prothrombin to thrombin
o Swiss chard o Cauliflower in a multistep hydrolytic process that
o Brussel sprouts o Parsley, fresh Prothrom- releases thrombin and a peptide fragment
o Broccoli binase called prothrombin fragment 1.2 (F 1.2).

→Vitamin K catalyzes an essential posttranslational Prothrombin Fragment 1.2

modification of the prothrombin group proteins: γ- →This is in plasma is thus a marker for
carboxylation of amino-terminal glutamic acids. thrombin generation.

Glutamic Acid COAGULATION COMPLEXES

→Modified to γ-carboxyglutamic acid when a second Complex Components Activates
carboxyl group is added to the γ carbon. →VIIa →IX and X
→Tissue Factor
γ-Carboxyglutamic Acid Extrinsic Tenase
→Phospholipid
→With two ionized carboxyl groups, it gains a net negative →Ca2+
charge, which enables them to bind ionic calcium (Ca2+). →IXa →X
→VIIa
→The bound calcium enables the vitamin K–dependent Intrinsic Tenase
→Phospholipid
proteins to bind to negatively charged phospholipids to → Ca2+
form coagulation complexes. →Xa →Prothrombin
→Va (II)
Vitamin K-Antagonism: des- γ-carboxyl Proteins Prothrombinase
→Phospholipid
→In vitamin K deficiency or in the presence of warfarin, a → Ca2+
vitamin K antagonist, the vitamin K–dependent
procoagulants are released from the liver without the
COFACTORS IN HEMOSTASIS
second carboxyl group added to the γ carbon.
Hemostatic Cofactors
→These are called des- γ -carboxyl proteins or proteins in
HEMOSTATIC COFACTORS
vitamin K antagonism (PIVKAs).
→Because they lack the second carboxyl group, they Cofactor Function Binds
cannot bind to Ca2+ and phospholipid, so they cannot Tissue Factor →Procoagulant →VIIa
participate in the coagulation reaction. V → Procoagulant →Xa
→Vitamin K antagonism is the basis for oral anticoagulant VIII → Procoagulant →IXa
(warfarin, Coumadin) therapy. High-Molecular- → Procoagulant →XIIa
→Vitamin K–dependent procoagulants are essential for the Weight →Pre-
assembly of three membrane complexes leading to the Fibrinogen kallikrein
generation of thrombin. (HMWK)
→Each complex is composed of a vitamin K–dependent →Coagulation →Thrombin
serine protease, its non-enzyme cofactor, and Ca2+, bound Control Factor:
to the negatively charged phospholipid membranes of Thrombomodulin Protein C
activated platelets or tissue factor–bearing cells. →Antifibrinolytic: →Thrombin
(TAFI)
THE 3 MEMBRANE COMPLEX: EXTRINSIC TENASE, →Coagulation →Protein C
Protein S
INTRINSIC TENASE, AND PROTHROMBINASE Control Factor →TFPI
→The initial complex that is composed of →Coagulation →ZPI
Protein Z
factor VIIa and tissue factor, and it Control Factor
activates factors IX and X, which are →Thrombomodulin is also a cofactor in control of
components of the next two complexes, fibrinolysis.
Extrinsic →Each cofactor binds its particular serine protease.
intrinsic tenase and prothrombinase,
Tenase →When bound to their cofactors, serine proteases gain
respectively.
stability and increased reactivity.
→Complex Composition: TF:VIIa Complex
→Factors Activated: Factor IX and X PROCOAGULANT COFACTORS
→It is composed of factor IXa and its →A transmembrane receptor for factor
cofactor VIIIa VIIa and is found on extravascular cells
→It also activates factor X much more Tissue such as fibroblasts and smooth muscle
Intrinsic
efficiently than the TF:VIIa complex. Factor cells, but under normal conditions, it is not
Tenase
(TF) found on blood vessel ECs.
→Complex Composition: Factor IXa and →Vessel injury exposes blood to the
Factor VIIa (cofactor) subendothelial tissue factor–bearing cells

40
1
and leads to activation of coagulation
through VIIa.
→Tissue factor is expressed in high levels
in cells of the:
o Brain
o Lung
o Placenta
o Heart
o Kidney
o Testes
→In inflammatory conditions and sepsis,
leukocytes and other cells can also express
tissue factor and initiate coagulation.
→A soluble plasma protein
→A glycoprotein circulating in plasma and
also present in platelet α-granules.
→During platelet activation and secretion,
platelets release partially activated factor
V at the site of injury.
→Factor Va is a cofactor to Xa in the
prothrombinase complex in coagulation.
Prothrombinase Complex:Va-Xa
→The prothrombinase complex
accelerates thrombin generation more
than 300,000-fold compared to Xa alone.
Factor V
Activation of Protein C by
Thrombomodulin-Bound Thrombin
→As described below, thrombomodulin-
bound thrombin activates protein C, which
inactivates Va to Vi.
→Therefore, factor V is both activated and
then ultimately inactivated by the
generation of thrombin, as is factor VIII.
! NOTE !
*Activated by thrombin
*Inactivated by protein C
→A soluble plasma protein.
→Factor VIII is a cofactor to factor IX,
which together form the intrinsic tenase
complex.
Factor VIII
! NOTE !
*Activated by thrombin
*Inactivated by protein C
→It is a cofactor to factor XIIa and
prekallikrein in the intrinsic contact factor
High-
complex, a mechanism for activating
Molecular-
coagulation in conditions where foreign
Weight
objects such as mechanical heart valves or
Kininogen
bacterial membranes and/or high levels of
inflammation are present.
CONTROL COFACTORS
→A transmembrane protein constitutively
expressed by vascular ECs, is a thrombin
Thrombo- cofactor.
modulin →Together, thrombomodulin and
thrombin activate protein C and thrombin
activatable fibrinolysis inhibitor (TAFI).
Protein C
→A coagulation regulatory protein.
Thrombin Activatable Fibrinolysis
Inhibitor (TAFI)
→A fibrinolysis inhibitor.
→In one of many examples of negative
feedback regulation in coagulation, once
thrombin is bound to thrombomodulin, it
loses its procoagulant ability to activate
factors V and VIII, and, through activation
of protein C, leads to destruction of factors
V and VIII, thus suppressing further
generation of thrombin.
→Both protein S and protein C are
cofactors in the regulation and control of
coagulation
! NOTE !
*Thrombomodulin loses its procoagulant
ability to activate factors V and VII once
thrombin is attached to it.
Protein S →A cofactor to protein C, as well as TFPI.
→A cofactor to Z-dependent protease
Protein Z
inhibitor (ZPI).
FACTOR VIII AND VON WILLEBRAND FACTOR
→Has a molecular mass of 260,000 Daltons and
is produced primarily by hepatocytes, but also
by microvascular ECs in lung and other tissues.
→Free factor VIII is unstable in plasma; it
circulates bound to VWF.
→During coagulation, thrombin cleaves factor
VIII from VWF and activates it.
Factor VIIIa
→Binds to activated platelets and forms the
intrinsic tenase complex with factor IXa and
Ca2+.
→Factor VIII and factor IX are the two plasma
procoagulants whose production is governed
by genes carried on the X chromosome.
Factor
VIII
Hemophilia
→Factor VIII deficiency
→Males with hemophilia A
Hemophilia
have diminished factor VIII
A
activity but normal VWF
levels.
Hemophilia →Factor IX deficiency
B
→These are sex-linked disorders occurring
almost exclusively in males.
→Factor VIII is a cofactor, but its importance in
hemostasis cannot be overstated, as evidenced
by the severe bleeding and symptoms
associated with hemophilia A.
→It deteriorates more rapidly than the other
coagulation factors in stored blood.
41
1

→In thawed component plasma, the factor VIII
level drops to approximately 50% after 5 days.
→Treatment for hemophilia bleeding episodes
consists of replacement therapy transfused
according to the 12-hour half-life of factor VIII.
→A large multimeric glycoprotein that
participates in platelet adhesion and transports
the procoagulant factor VIII.
→It is composed of multiple subunits of
240,000 Daltons each.
→The subunits are produced by ECs and
megakaryocytes, where they combine to form
multimers that range from 600,000 to
20,000,000 Daltons.
Storage of VWF
→VWF molecules are stored in α-granules in
platelets and in Weibel-Palade bodies in ECs.
→The molecules are released from storage into
the plasma, and they circulate at a
concentration of 7 to 10 mg/mL.
→ECs release ultralarge multimers of VWF into
plasma, where they are normally degraded into
smaller multimers by a VWF-cleaving protease,
ADAMTS-13, in blood vessels with high shear
stress.
ADAMTS-13
→A disintegrin and metalloproteinase with a
thrombospondin type 1 motif, member 13
Thrombotic Thrombocytopenic Purpura (TTP)
→Inherited or acquired defective ADAMTS-13
VWF enzyme activity is associated with the presence
of ultralarge VWF multimers in plasma,
resulting in platelet aggregation and
microvascular thrombosis.
→VWF has receptor sites for both platelets and
collagen and helps to bind platelets to exposed
subendothelial collagen during platelet
adhesion, especially in arteries and arterioles
where the flow of blood is faster.
→The primary platelet surface receptor for
VWF is GP Ib/IX/V.
→Arginineglycine-aspartic acid (RGD)
sequences in VWF also bind a second platelet
integrin, GP IIb/IIIa, during platelet
aggregation.
→A third site on the VWF molecule binds
collagen, and a fourth site binds the plasma
procoagulant cofactor, factor VIII.
PLATELET MEMBRANE BINDING SITES OF
VWF
→GP Ib/IX/V: Primary platelet
1st Site surface receptor for platelet
adhesion
→GP IIb/IIIa: Platelet integrin for
2nd Site
platelet aggregation
3rd Site →Collagen I and II
→Factor VIII: Procoagulant
4th Site
Cofactor
Von Willebrand Disease
→VWF is decreased in von Willebrand disease
(VWD)
→A relatively common disorder that occurs in
1% to 2% of the general population.
→Because factor VIII depends on VWF for
stability, individuals with VWD who have
diminished VWF also have diminished factor
VIII activity levels.
→Typically, factor VIII levels decrease to
hemorrhagic levels (less than 30%) only in
severe VWD.
ABO Blood Type: Group O
→The level of VWF also varies in people
according to their ABO blood type.
→Group O individuals have lower levels of
VWF than other ABO types.
! NOTE !
*VWF and Factor VIII is an acute phase protein and their
levels increase in:
o Pregnancy
o Trauma
o Infections
o Stress
FACTOR XI AND THE CONTACT FACTORS
→Also called intrinsic accessory pathway
proteins, are:
o Factor XII
o High-molecular-weight kininogen
(HMWK) or Fitzgerald Factor
o Prekallikrein (pre-K) orFletcher
Factor
→They are so named because they are
activated by contact with negatively charged
foreign surfaces.
Factor XIIa
→Transforms pre-K, a glycoprotein that
circulates bound to HMWK, into its active form
kallikrein, which cleaves HMWK to form
bradykinin.
Contact
Factors Factor XII and Pre-K
→These are zymogens that are activated to
become serine proteases
HMWK
→A nonenzymatic cofactor.
Contact Factor Complex: HMWK:pre-K:FXII)
→Activates factor XI
→Factor XIa is an activator of factor IX.
→Deficiencies of factor XII, HMWK, or pre-K do
not cause clinical bleeding disorders.
→However, deficiencies do prolong laboratory
tests and necessitate investigation.
Factor XII

42
1

→It is activated in vitro by negatively charged →The primary substrate of thrombin, which
surfaces such as: converts soluble fibrinogen to insoluble
o Nonsiliconized glass fibrin to produce a clot.
o Kaolin →It is also essential for platelet aggregation
o Ellagic acid in partial thromboplastin because it links activated platelets through
time (PTT) reagents their GP IIb/IIIa platelet fibrinogen receptor.
→It is a 340,000 Dalton glycoprotein
→In vivo, foreign materials such as stents or synthesized in the liver.
valve prostheses may activate contact factors to →The normal plasma concentration of
cause thrombosis. fibrinogen ranges from 200 to 400 mg/dL,
→It is activated by the contact factor complex the most concentrated of all the plasma
and, more significantly, by thrombin during procoagulants.
coagulation generated from tissue factor →It is an acute phase reactant protein, whose
activation. level increases in inflammation, infection,
and other stress conditions.
Factor XIa
→Activates factor IX Platelet α-granules
Factor
→Absorb, transport, and release abundant
XI
Rosenthal Syndrome fibrinogen.
→Deficiencies of factor XI usually result in mild
and variable bleeding. →The fibrinogen molecule is a mirror-image
→Factor XI supplements or boosts factor IX “trinodular” dimer, each half consisting of
activation, so deficiencies of factor XI are less three nonidentical polypeptides, designated
Fibrinogen
severe clinically than deficiencies of the other Aα, Bβ, and γ, united by disulfide bonds.
(FI)
factors such as IX or VIII.
THROMBIN (FIIa) E Domain
Primary Function →A bulky central region that is formed by
→Cleave fibrinopeptides A and B from the α and β chains the assembly of the six N-terminals.
of the fibrinogen molecule, triggering spontaneous fibrin
polymerization. D Domain
→The carboxyl terminals assemble at the
→It also amplifies the coagulation mechanism by activating outer ends of the molecule to form two D
cofactors V and VIII and factor XI by a positive feedback domains.
mechanism.
→It also activates factor XIII, which forms covalent bonds →Thrombin cleaves fibrinopeptides A and B
between the D domains of the fibrin polymer to cross-link from the protruding N-termini of each of the
and stabilize the fibrin clot. two α and β chains of fibrinogen, reducing
→It also initiates aggregation of platelets. the overall molecular weight by 10,000
→Thrombin bound to thrombomodulin activates the Daltons.
protein C pathway to suppress coagulation, and it activates
TAFI to suppress fibrinolysis. Fibrin Monomer
→Summary of where it plays a role: →Cleaved fibrinogen
o Coagulation (Fibrin) →The exposed fibrin monomer α and β chain
o Platelet activation ends (E domain) have an immediate affinity
o Coagulation control (Protein C) for portions of the D domain of neighboring
o Controlling fibrinolysis (TAFI). monomers, spontaneously polymerizing to
form fibrin polymer.
→Because of its multiple autocatalytic functions, thrombin →A heterodimer whose subunits are:
is considered the key protease of the coagulation pathway. →Produced mostly by
α subunit megakaryocytes and
! NOTE ! monocytes
*Thrombin + Thrombomodulin = β subunit →Produced in the liver.
o Suppressed Coagulation due to Protein C
o Suppressed Fibrinolysis due to TAFI Factor XIIIa
*Activated Factors by Thrombin: →Covalently crosslinks fibrin polymers to
Factor XIII
o V form a stable insoluble fibrin clot.
o VIII →A transglutaminase that catalyzes the
o XI formation of covalent bonds between the
o XIII carboxyl terminals of γ chains from adjacent
FIBRINOGEN (FI) STRUCTURE AND FIBRIN FORMATION, D domains in the fibrin polymer.
FACTOR XIII →These bonds link the ε-amino acid of lysine
moieties and the γ-amide group of glutamine
units.

43
1
→Multiple cross-links form to provide an
insoluble meshwork of fibrin polymers linked
by their D domains, providing physical
strength to the fibrin clot.
→Factor XIIIa reacts with other plasma and
cellular structural proteins and is essential to
wound healing and tissue integrity.
→Cross-linking of fibrin polymers by factor
XIIIa covalently incorporates fibronectin, a
plasma protein involved in cell adhesion, and
α2-antiplasmin, rendering the fibrin mesh
resistant to fibrinolysis.
Plasminogen
→The primary serine protease of the
fibrinolytic system, also becomes covalently
bound via lysine moieties, as does TPA
TPA
→A serine protease that ultimately
hydrolyzes and activates bound plasminogen
to initiate fibrinolysis.
PLASMA-BASED (IN VITRO) COAGULATION: EXTRINSIC,
INTRINSIC, AND COMMON PATHWAYS
→In the past, two coagulation pathways were described,
both of which activated factor X at the start of a common
pathway leading to thrombin generation.
→The pathways were characterized as cascades in that as
one enzyme became activated, it in turn activated the next
enzyme in sequence.
→Most coagulation experts identified the activation of
factor XII as the primary step in coagulation because this
factor could be found in blood, whereas tissue factor could
not.
→Consequently, the reaction system that
begins with factor XII and culminates in fibrin
polymerization has been called the intrinsic
pathway.
→The coagulation factors of the intrinsic
pathway, in order of reaction, are:
o Factors XII, XI, IX, VIII, X, V, and
prothrombin (II), and fibrinogen (I)
o Pre-K
o HMWK
Order of Activation
Factor XII → pre-K→ HMWK → XI → IX → VIII
Intrinsic
→ X → V → Prothrombin (FII) → Fibrinogen
Pathway
(FI)
→The laboratory test that detects the absence
of one or more of these factors is the activated
partial thromboplastin time (APTT or PTT)
Deficiency of Contact Factors = Longer PTT
→We now know that the contact factors XII,
pre-K, and HMWK do not play a significant role
in in vivo coagulation with trauma-type
injuries, although their deficiencies prolong the
in vitro laboratory tests of the intrinsic
pathway, in particular, the PTT.
→Formation of TF:VIIa has since proven to be
the primary in vivo initiation mechanism for
coagulation.
→Because tissue factor is not present in blood,
the tissue factor pathway has been called the
extrinsic pathway.
→This pathway includes the factors:
o VII
o X
o V
o Prothrombin (II)
o Fibrinogen (I)
Order of Activation
TF:VIIa→ X → V → Prothrombin (II)
→Fibrinogen (I)
Extrinsic
Pathway Prothrombin Time Test (PT)
→The test used to measure the integrity of the
extrinsic pathway.
→The PT and PTT are assays often used in
tandem to screen for coagulation factor
deficiencies.
→Factor VIII and factor IX are not considered
to be part of the extrinsic pathway, because the
PT fails to identify their absence or deficiency.
→But clearly, the IXa:VIIIa complex in the
intrinsic pathway is crucial to the activation of
factor X.
→Deficiencies of either one of these
components—factor VIII in hemophilia A, or
factor IX in hemophilia B—can result in severe
and life-threatening hemorrhage.
→The two pathways have in common:
o Factor X
o Factor V
Common
o Prothrombin
Pathway
o Fibrinogen
→This portion of the coagulation pathway is
often called the common pathway.
→These designations—intrinsic, extrinsic, and common—
are used extensively to interpret in vitro laboratory testing
and to identify factor deficiencies
→However, they do not adequately describe the complex
interdependent reactions that occur in vivo.
CELL-BASED (IN VIVO, PHYSIOLOGIC) COAGULATION
→An intricate combination of cellular and biochemical
events function in harmony to keep blood liquid within the
veins and arteries, to prevent blood loss from injuries by
the formation of thrombi, and to reestablish blood flow
during the healing process.
→As noted above, the series of cascading proteolytic
reactions traditionally known as the extrinsic and intrinsic
coagulation pathways do not fully describe how
coagulation occurs in vivo.
→These pathways are not distinct, independent,
alternative mechanisms for generating thrombin but are
actually interdependent.
EXAMPLES THAT SHOWS INTERDEPENDENCE OF
INTRINSIC AND EXTRINSIC PATHWAY
44
1
→A deficiency of factor VII in the extrinsic
1 pathway can cause significant bleeding, even when
the intrinsic pathway is intact.
→Deficiencies of factors VIII and IX may cause
2 severe bleeding, regardless of the presence of a
normal extrinsic pathway.
→In addition to procoagulant and anticoagulant plasma
proteins, normal physiologic coagulation requires the
presence of two cell types for formation of coagulation
complexes:
o Cells that express tissue factor (usually
Extravascular)
o Platelets (Intravascular)
2 OCCURING PHASES OF COAGULATION
→Occurs on tissue factor–expressing
Initiation cells and produces 3% to 5% of the total
thrombin generated
→Occurring on platelets, which produces
Propagation
95% or more of the total thrombin
INITIATION
→In vivo, the principle mechanism for generating
thrombin is begun by formation of the extrinsic tenase
complex, rather than the intrinsic pathway.
Initiation Phase
→Refers to extrinsic tenase complex formation and
generation of small amounts of factor Xa, factor IXa, and
thrombin.
→Damage to the endothelium spills blood and
platelets into the extravascular tissue and triggers
a localized response.
→The magnitude of the response depends largely
1 on the:
o Extent of the injury
o How large the bleed is
o How much tissue is damaged
o How many platelets are available
→About 1% to 2% of factor VIIa is present
normally in blood in the activated form, but it is
inert until bound to tissue factor and is unaffected
2
by TFPI and other inhibitors.
→Fibroblasts and other subendothelial cells
provide tissue factor, a cofactor to factor VIIa.
→Factor VIIa binds to tissue factor on the
3 membrane of subendothelial cells, and the
extrinsic tenase complex TF:VIIa is formed.
TF:VIIa: Extrinsic Tenase Complex
4 →TF:VIIa activates low levels of both factor IX and
factor X.
Membrane-Bound Xa and Prothrombinase
Complex Xa:Va
5 →Minute amounts of thrombin are generated by
membrane-bound Xa and Xa:Va prothrombinase
complexes.
Factor Va
→Factor Va comes from the activation of plasma
6
factor V by thrombin, by platelets if there has been
an injury, or by noncoagulation proteases.
→Coagulation complexes bound to cell membranes are
relatively protected from inactivation by most inhibitors.
Xa:Va Dissociation
→However, if Xa:Va dissociates from the cell, it is rapidly
inactivated by the protease inhibitors TFPI, antithrombin,
and protein Z–dependent protease inhibitor (ZPI) until a
threshold of Xa:Va activity is reached.
RESULTS OF INITIATION PHASE DESPITE THE MINUTE
FORMATION OF THROMBIN
→Platelets, cofactors, and procoagulants become
1
activated
2 →Fibrin formation begins
3 →Initial platelet plug is formed
FUNCTION OF THE MINUTE THROMBIN PRODUCED IN
THE INITIATION PHASE
→Activates platelets through cleavage of protease
1
activated receptors PAR-1 and PAR-4
→Activates factor V released from platelet α-
2
granules
3 →Activates factor VIII and dissociates it from VWF
→Activates factor XI, the intrinsic accessory
4
procoagulant that activates more factor IX
→Splits fibrinogen peptides A and B from
5 fibrinogen and forms a preliminary fibrin
network.
Cleavage of Fibrinopeptides
→Occurs at the end of the initiation phase and beginning of
the propagation phase.
→In most clot-based coagulation assays, this is the visual
endpoint of the assay.
→It occurs with only 10 to 30 nmol/L of thrombin, or
approximately 3% of the total thrombin generated.
PROPAGATION
→More than 95% of thrombin generation occurs during
propagation.
→In this phase the reactions occur on the surface of the
activated platelet, which now has all the components
needed for coagulation.
→Large numbers of platelets adhere to the site of injury,
localizing the coagulation response.
→Platelets are activated at the site of injury by both the
low-level thrombin generated in the initiation phase and by
adhering to exposed collagen.
COAT-Platelets
→Platelets partially activated by collagen and thrombin.
→These partially activated COAT-platelets have a higher
level of procoagulant activity than platelets exposed to
collagen alone.
→They also provide a surface for formation and
amplification of intrinsic tenase and prothrombinase
complexes.
→The cofactors Va and VIIIa activated by
1 thrombin in the initiation phase bind to platelet
membranes and become receptors for Xa and IXa.
2 IXa:VIIIa: Intrinsic Tenase Complex
45
1
→IXa generated in the initiation phase binds to
VIIIa on the platelet membrane to form the
intrinsic tenase complex IXa:VIIIa.
→More factor IXa is also generated by platelet-
bound factor XIa.
→This intrinsic tenase complex activates factor X
3 at a 50- to 100-fold higher rate than the extrinsic
tenase complex.
Xa:Va: Prothrombinase Complex
→Factor Xa binds to Va to form the
4
prothrombinase complex, which activates
prothrombin and generates a burst of thrombin.
→Thrombin cleaves fibrinogen into a fibrin clot,
activates factor XIII to stabilize the clot, binds to
thrombomodulin to activate the protein C control
5 pathway, and activates TAFI to inhibit fibrinolysis.
→Since coagulation depends on the presence of
both tissue factor–bearing cells and activated
platelets, clotting is localized to the site of injury.
→Protease inhibitors and intact endothelium
6 prevent clotting from spreading to other parts of
the body.
→It may be helpful operationally to think of the extrinsic or
tissue factor pathway as occurring on the tissue factor
bearing cell and the intrinsic pathway (minus factors XII,
HMWK, and pre-K) as occurring on the platelet surface.
→However, these are not separate and redundant
pathways; they are interdependent and occur in parallel
until blood flow has ceased and termination by control
mechanisms takes place.
→Both platelets and tissue factor–bearing cells are
essential for physiologic coagulation.
→Deficiencies of any of the key proteins of coagulation
complex formation and activity (VII, IX, VIII, X, V, or
prothrombin) compromise thrombin generation and
manifest as significant bleeding disorders.
COAGULATION REGULATORY MECHANISMS
(1) Coagulation Regulatory Mechanisms
(2) Tissue Factor Pathway Inhibitor
(3) Protein C Regulatory System
(4) Antithrombin and Other Serine Protease Inhibitors
(Serpins)
COAGULATION REGULATORY MECHANISMS
→Inhibitors and their cofactors regulate serine proteases
and cofactors in the coagulation system.
→They also provide feedback loops to maintain a complex
and delicate balance between thrombosis and abnormal
bleeding.
Inhibitors/Natural Anticoagulants
→Function to slow the activation of procoagulants and
suppress thrombin production.
→They ensure that coagulation is localized and is not a
systemic response, and they prevent excessive clotting or
thrombosis.
PRINCIPAL COAGULATIONREGULATORS
1 →Tissue Factor Pathway Inhibitor (TFPI)
2 →Antithrombin (AT)
→Activated Protein C (From endpoint of protein C
3
pathway)
→Acquired or inherited deficiencies of these proteins may
be associated with increased incidence of venous
thromboembolic disease, as the hemostatic balance is
shifted more toward coagulation than termination of the
activated pathway.
TISSUE FACTOR PATHWAY INHIBITOR (TFPI)
→A Kunitz-type serine protease inhibitor and is the
principal regulator of the tissue factor pathway.
→The Kunitz-2 domain binds to and inhibits factor Xa, and
Kunitz-1 binds to and inhibits VIIa:TF.
→TFPI is synthesized primarily by ECs and is also
expressed on platelets.
→In the initiation of coagulation, factor VIIa and tissue
factor combine to activate factors IX and X.
→TFPI inhibits coagulation in a two-step process by first
binding and inactivating Xa.
2 STEP PROCESS OF TFPI INHIBITION OF
COAGULATION
Binding and Inactivation of Xa
1
→Formation of TFPI:Xa complex
Formation of Quaternary Complex
→TFPI:Xa complex then binds to TF:VIIa, forming
2
a quaternary complex and preventing further
activation of X and IX.
ALTERNATE TFPI INHIBITION OF COAGULATION
→ TFPI may bind to Xa in the TF:VIIa:Xa complex and
inactivate Xa and TF:VIIa.
→TFPI provides feedback inhibition, because it is not
actively engaged until coagulation is initiated and factor X
is activated.
Protein S
→Cofactor of activated protein C (APC) and TFPI
→Enhances factor Xa inhibition by TFPI tenfold.
→Because of the inhibitory action of TFPI, the TF:VIIa:Xa
reaction is short-lived.
→Once TFPI shuts down extrinsic tenase and Xa, additional
Xa and IXa production shifts to the intrinsic pathway.
→Propagation of coagulation occurs as factor X is activated
by IXa:VIII and more factor IX is activated by factor XIa.
PROTEIN C REGULATORY SYSTEM
→During coagulation, thrombin propagates the clot as it
cleaves fibrinogen and activates factors V, VIII, XI, and XIII.
→In intact normal vessels, where coagulation would be
inappropriate, thrombin avidly binds the EC membrane
protein thrombomodulin and triggers an essential
coagulation regulatory system called the protein C
anticoagulant system.
Protein C System
→It revises thrombin’s function from a procoagulant
enzyme to an anticoagulant.
Endothelial Cell Protein C Receptor (EPCR)
→EC protein C receptor (EPCR) is a transmembrane
protein that binds both protein C and APC adjacent to the
thrombomodulin-thrombin complex and augments the
46
1
action of thrombin-thrombomodulin at least fivefold in
activating protein C to a serine protease.
Activated Protein C (APC)
→It dissociates from EPCR and binds its cofactor, free
plasma protein S.
o Free Plasma Protein S: Cofactor of ACP
ACP-Protein S Complex
→The stabilized APC-protein S complex hydrolyzes and
inactivates factors Va and VIIIa, slowing or blocking
thrombin generation/coagulation.
Protein S
→The cofactor that binds and stabilizes APC, is synthesized
in the liver and circulates in the plasma in two forms.
2 FORMS OF PROTEIN S IN THE PLASMA
Free Protein S
1 →40% of protein S is free
→Serve as the APC cofactor
Complement Control Protein C4b-Binding Protein
(C4bBP)
→60% is covalently bound to the complement
2
control protein C4b-binding protein (C4bBP).
→Bound protein S cannot participate in the protein
C anticoagulant pathway
Protein S-C4bBP Binding
→It is of particular interest in inflammatory conditions
because C4Bbp is an acute phase reactant.
→When the plasma C4bBP level increases, additional
protein S is bound, and free protein S levels become
proportionally decreased, which may increase the risk of
thrombosis.
! NOTE !
*C4bBP: Bound Protein S:  Free Protein S
Recurrent Venous Thromboembolic Disease
→Chronic acquired or inherited protein C or protein S
deficiency or mutations of protein C, protein S, or factor V
compromise the normal downregulation of factors Va and
VIIIa and may be associated with recurrent venous
thromboembolic disease.
Purpura Fulminans
→Underscoring the importance of the protein C regulatory
system, neonates who completely lack protein C have a
massive thrombotic condition called purpura fulminans
and die in infancy unless treated with protein C
replacement and anticoagulation.
ANTITHROMBIN AND OTHER SERINE PROTEASE
INHIBITORS (SERPINS)
Antithrombin (AT)
→It was the first of the coagulation regulatory proteins to
be identified and the first to be assayed routinely in the
clinical hemostasis laboratory.
OTHER MEMBERS OF THE SERPIN FAMILY
1 →Heparin Cofactor II
2 →Protein Z-dependent Protease Inhibitor (ZPI)
3 →Protein C Inhibitor
4 →α1-protease inhibitor (α1-antitrypsin)
5 →α2-macroglobulin
6 →α2-antiplasmin
7 →PAI-1
Antithrombin
→A serine protease inhibitor (serpin) that binds and
neutralizes serine proteases, including:
o Thrombin (Factor IIa)
o Factors IXa, Xa, XIa, XIIa
o Prekallikrein
o Plasmin
Heparin Cofactor II
→A serpin that primarily inactivates thrombin.
→AT and heparin cofactor II both require heparin for
effective anticoagulant activity.
Heparin
→In vivo, heparin is available from endothelium associated
mast cell granules or as EC heparan sulfate, a natural
glycosaminoglycan that activates AT, although not to the
same intensity as therapeutic unfractionated heparin.
→AT’s activity is accelerated 2000-fold by binding to
heparin and is the basis for the anticoagulant activity of
pharmaceutical heparin.
→Therapeutically, heparin is administered as either of the
following:
→Consists of chains of greater than
18 sugar units and accelerates
Unfractionated
inactivation of thrombin through
Heparin
heparin-dependent conformational
changes and bridging mechanisms.
Low-Molecular- →Lacking long polysaccharide
Weight Heparin chains for thrombin inactivation, AT
Heparin preferentially inactivates factor Xa
Pentasaccharide
Inactive Thrombin-Antithrombin Complex (TAT)
→In vivo, antithrombin covalently binds thrombin, forming
an inactive thrombin-antithrombin complex (TAT), which
is then released from the heparin molecule.
→Laboratory measurement of TAT is used as an indicator
for thrombosis, since it measures both the generation of
thrombin and its inhibition.
Antithromb
in
Heparin
Cofactor II
→ZPI, in the presence of its cofactor, protein
Z, is a potent inhibitor of factor Xa.
Protein Z- ZPI
Dependent →It covalently binds protein Z and factor Xa
Protease in a complex with Ca2+ and phospholipid.
Inhibitor →It also inhibits factor XIa, in a separate
(ZPI) reaction that does not require protein Z,
phospholipid, and Ca2+.
→The inhibition of factor XIa is accelerated
twofold by the presence of heparin.
47
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Protein Z
→A vitamin K–dependent plasma
glycoprotein that is synthesized in the liver.
→Although protein Z has a structure similar
to that of the other vitamin K–dependent
proteins (factors II, VII, IX, and X and protein
C), it lacks an activation site and, like
protein S, is nonproteolytic.
→It increases the ability of ZPI to inhibit
factor Xa 2000-fold.
→It is a nonspecific, heparin-binding serpin
that inhibits a variety of proteases,
including:
o APC
o Thrombin
o Factor Xa and Xia
o Urokinase
→It is found not only in plasma but also in
many other body fluids and organs.
Protein C
Inhibitor FUNCTION BASED ON TARGET
Anticoagula →Inhibits thrombin
nt
→Inhibits thrombin-
Procoagula
thrombomodulin
nt
and APC
Fibrinolytic
Inhibitor

α1-protease →Serpins that are able to inhibit serine

inhibitor proteases reversibly.
and α2-
macro-
globulin

COAGULATION REGULATORY MECHANISMS: COAGULATION REGULATORY PROTEINS

48
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ANTITHROMBIN AND OTHER SERINE PROTEASE INHIBITORS (SERPINS): PROTEINS OF THE FIBRINOLYSIS PATHWAY
FIBRINOLYSIS
(1) Fibrinolysis
(2) Plasminogen and Plasmin
(3) Plasminogen Activation
(a) Tissue Plasminogen Activator (TPA)
(b) Urokinase Plasminogen Activator (UPA)
(4) Control of Fibrinolysis
(a) Plasminogen Activator Inhibitor 1 (PAI-1)
(b) α2-Antiplasmin
(c) Thrombin-Activatable Fibrinolysis Inhibitor
(5) Fibrin Degeneration Products and D-Dimer
→Fibrinolysis, the final stage of coagulation, begins a few
hours after fibrin polymerization and cross-linking.
2 Activators of Fibrinolysis: Tissue Plasminogen Activator
(TPA) and Urokinase Plasminogen Activator (UPA)
→These are released in response to inflammation and
coagulation.
→Activate fibrin-bound plasminogen several hours after
thrombus formation, degrading fibrin and restoring normal
blood flow during vascular repair.
→Fibrinolytic proteins assemble on fibrin during clotting.
→Plasminogen, plasmin, TPA, UPA, and PAI-1 become
incorporated into the fibrin clot as they bind to lysine through
their “kringle” loops, thereby concentrating and localizing
them to the fibrin clot.
→Fibrinolysis is the systematic, accelerating hydrolysis of
fibrin by bound plasmin.
→Again, there is a delicate balance between activators and
inhibitors.
Excessive →Can cause bleeding due to premature clot
Fibrinolysis lysis before wound healing is established
Inadequate →Can lead to clot extension and
Fibrinolysis thrombosis.
PLASMINOGEN AND PLASMIN
Plasminogen
→A 92,000 Dalton plasma zymogen produced by
the liver.
→It is a single-chain protein possessing five glycosylated
loops termed kringles.
Kringles
→Enable plasminogen, along with activators TPA and UPA,
to bind fibrin lysine molecules during polymerization.
→This fibrin-binding step is essential to fibrinolysis.
→Fibrin-bound plasminogen becomes converted into a
two-chain active plasmin molecule when cleaved between
arginine at position 561 and valine at position 562 by
neighboring fibrin-bound TPA or UPA.
Plasmin
→A serine protease that systematically digests fibrin
polymer by the hydrolysis of arginine-related and lysine-
related peptide bonds.
Bound Plasmin
→Digests clots and restores blood vessel patency.
→Its localization to fibrin through lysine binding prevents
systemic activity.
→As fibrin becomes digested, the exposed carboxy-
terminal lysine residues bind additional plasminogen and
TPA, which further accelerates clot digestion.
Free Plasmin
→Capable of digesting plasma fibrinogen, factor V, factor
VIII, and fibronectin, causing a potentially fatal primary
fibrinolysis.
→However, plasma a2-antiplasmin rapidly binds and
inactivates any free plasmin in the circulation.
PLASMINOGEN ACTIVATION
→ECs secrete TPA, which hydrolyzes
Tissue
fibrin-bound plasminogen and initiates
Plasminogen
fibrinolysis.
Activator
→TPA, with two glycosylated kringle
(TPA)
regions, forms covalent lysine bonds with
49
1
fibrin during polymerization and localizes
at the surface of the thrombus with
plasminogen, where it begins the
digestion process by converting
plasminogen to plasmin.
→Circulating TPA is bound to inhibitors
such as PAI-1 and is cleared from plasma.
→Synthetic recombinant TPAs mimic
intrinsic TPA and are a family of drugs
used to dissolve pathologic clots that form
in venous and arterial thrombotic disease.
→Urinary tract epithelial cells, monocytes,
and macrophages secrete another
intrinsic plasminogen activator called
urokinase plasminogen activator.
→UPA circulates in plasma at a
concentration of 2 to 4 ng/mL and
Urokinase becomes incorporated into the mix of
Plasminongen fibrin-bound plasminogen and TPA at the
Activator time of thrombus formation.
(UPA) →UPA has only one kringle region, does
not bind firmly to fibrin, and has a
relatively minor physiologic effect.
→Like TPA, purified UPA preparations are
used to dissolve thrombi in myocardial
infarction, stroke, and deep vein
α2-
thrombosis.
Antiplasmin
CONTROL OF FIBRINOLYSIS
→The principal inhibitor of plasminogen
activation, inactivating both TPA and UPA
and thus preventing them from converting
plasminogen to the fibrinolytic enzyme
plasmin.
→A single-chain glycoprotein serine
protease inhibitor
CELL TYPES THAT PRODUCE PAI-1
→ECs →Monocytes
→Megakaryocytes →Adipocytes
→Smooth muscle →Hepatocytes
cells
→Fibroblasts →Other cell types
Plasminogen
Activator →Platelets store a pool of PAI-1,
Inhibitor 1 accounting for more than half of its
(PAI-1) availability and for its delivery to the
fibrin clot.
→It is present in excess of the TPA
concentration in plasma, and circulating
Thrombin-
TPA normally becomes bound to PAI-1.
Activatable
→Only at times of EC activation, such as
Fibrinolysis
after trauma, does the level of TPA
Inhibitor
secretion exceed that of PAI-1 to initiate
fibrinolysis.
→Binding of TPA to fibrin protects TPA
from PAI-1 inhibition.
→Plasma PAI-1 levels vary widely.
PAI-1 Deficiency
→It has been associated with chronic mild
bleeding due to increased fibrinolysis.
→PAI-1 is an acute phase reactant and is
increased in many conditions, including:
o Metabolic syndrome
o Obesity
o Atherosclerosis
o Sepsis
o Stroke.
! NOTE !
Increased PAI-1 levels correlate with
reduced fibrinolytic activity and increased
risk of thrombosis.
→It is synthesized in the liver and is the
primary inhibitor of free plasmin.
→AP is a serine protease inhibitor with
the unique characteristic of both N- and C-
terminal extensions.
→During thrombus formation, the N
terminus of AP is covalently linked to
fibrin by factor XIIIa.
→The C-terminal contains lysine, which is
capable of lysine-binding kringles of
plasmin.
→Free plasmin produced by activation of
plasminogen can bind either to fibrin,
where it is protected from AP because its
lysine-binding site is occupied, or to the C-
terminus of AP, which rapidly and
irreversibly inactivates it.
→Thus AP with its C-terminal lysine slows
fibrinolysis by competing with lysine
residues in fibrin for plasminogen binding
and by binding directly to plasmin and
inactivating it.
→The therapeutic lysine analogues,
tranexamic acid and e-aminocaproic acid,
are similarly antifibrinolytic through their
affinity for kringles in plasminogen and
TPA.
→Both inhibit the proteolytic activity of
plasmin.
→It is a plasma procarboxypeptidase
synthesized in the liver that becomes
activated by the thrombin-
thrombomodulin complex.
→This is the same complex that activates
the protein C pathway; however, the two
functions are independent.
Activated TAFI
→Functions as an antifibrinolytic enzyme.
→It inhibits fibrinolysis by cleaving
exposed carboxy-terminal lysine residues
from partially degraded fibrin, thereby
preventing the binding of TPA and
plasminogen to fibrin and blocking the
formation of plasmin.
Coagulation Factor-Deficient States
→In these cases, such as hemophilia,
decreased thrombin production may
reduce the activation of TAFI, resulting in
50
1

increased fibrinolysis that contributes to

more bleeding.

Thrombotic Disorders
→In this cases, increased thrombin
generation may increase the activation of
TAFI.
→The resulting decreased fibrinolysis
may contribute further to thrombosis.

→TAFI also may play a role in regulating

inflammation and wound healing.
FIBRIN DEGENERATION PRODUCTS AND D-DIMER
→Plasmin cleaves fibrin and produces a series of
identifiable fibrin fragments: X, Y, D, E, and D-D.
→Several of these fragments inhibit hemostasis and
contribute to hemorrhage by preventing platelet activation
and by hindering fibrin polymerization.

→Described as the central E domain with

Fragment X the two D domains (D-E-D), minus some
peptides cleaved by plasmin.
→The E domain after cleavage of one D
Fragment Y
domain (D-E).
→It is composed of two D domains from
separate fibrin molecules cross-linked by
the action of factor XIIIa.

Fragments X, Y, D, and E
→Produced by digestion of either fibrin
D-D
or fibrinogen by plasmin
Fragment:
D-Dimer
Production of D-Dimer
→D-dimer is a specific product of
digestion of cross-linked fibrin only and
is therefore a marker of thrombosis and
fibrinolysis—that is, thrombin, factor
XIIIa, and plasmin activation.
→Eventually these fragments (X and Y) are further
digested to individual D and E domains.

→The various fragments may be detected by quantitative

or semiquantitative immunoassay to reveal fibrinolytic
activity.
→D-dimer is separately detectable by monoclonal antibody
for D-dimer antigen, using a wide variety of automated
quantitative laboratory immunoassays and other formats
including point-of-care tests performed on whole blood.

D-Dimer Immunoassay
→It is used to identify chronic and acute DIC and to rule
out venous thromboembolism in suspected cases of deep
venous thrombosis or pulmonary embolism.

51