BIOCHEMISTRY

HEMOSTASIS AND THROMBOSIS

Marie Aileen Frances M. Carlos, M.D. || 09/20/2017

OUTLINE
D. Cofactors
I. Circulatory System
E. Regulation
II. Hemostasis
V. Fibrinolysis
A. Definition
A. Definition
B. Phases of Hemostasis
B. Clot Busters
1. Primary
VI. Anticoagulant
2. Secondary
A. Definition
3. Fibrinolysis
B. Function
III. Platelet
C. Four Major Classes
A. Definition
D. Three Major Classes of
B. Function
Drugs that Inhibit Blood
C. Platelet Plug Formation
Coagulation
1. Adhesion
VII. Disorders
2. Activation
A. Hereditary Coagulation
3. Aggregation
Disorders
IV. Coagulation Cascade
B. Pharmacological
A. Overview
Intervention in Bleeding
B. Nomenclature of Blood
VIII. Blood Coagulation Test
Clotting Factors
A. Platelet Assays
C. Pathways of Blood
B. Blood Clotting
Coagulation
Coagulation Factors Assays

OBJECTIVES
1. Explain the process of hemostasis
a. Role of platelets in hemostasis
2. Discuss the coagulation cascade:
a. Intrinsic and Extrinsic Pathways
b. Regulation-Activators and Inhibitors
c. Fibrinolysis-Activators and Inhibitors
3. Relate the action of anticoagulants
4. Explain the Biochemical basis of coagulation disorders
I. CIRCULATORY SYSTEM
 Efficient system used for delivery of nutrients to tissues
 Blood circulation through the endothelium of the vessels
 Prone to leakage 2nd degree to endothelial surface damage
II. HEMOSTASIS
A. DEFINITION
 Cessation of bleeding from a cut or a severed vessel (Harper’s)
 Coordinated function of blood vessels, platelets, coagulation
factors, and fibrinolytic system
 There is initial vasoconstriction of the injured blood vessel
Thrombosis
 Involves the formation of a blood clot or thrombus inside a
blood vessel.
 The degree or intensity is comparatively higher, and this
usually occurs due to an underlying health condition.
 The clot formed this way can obstruct the blood flow to the
affected site and can lead to serious complications if it
moves to vessels which supply blood to the important
organs in the body such as brain or lungs.
 Can be venous or arterial
 Caused by: hypercoagulability, injury to blood vessel walls
and venous stasis
Figure 1. Overview of the Phases of Hemostasis
(source: https://www.dreamstime.com/stock-illustration-basic-steps-hemostasis-vector-
diagram-image53475140)
B. PHASES OF HEMOSTASIS
1. Primary
 In the presence of damage to a blood vessel, platelets
immediately begin to adhere to the cut edges of the vessel
and release chemicals to attract even more platelets
 Temporarily forming a plug
 Cessation of external bleeding
 Formation of a loose and temporary platelet aggregate at the
site of injury
 Platelets bind to collagen at the site of vessel wall injury
and form thromboxane A2, and release ADP, which
activates other platelets flowing by the vicinity of the injury.
 Thrombin, formed during coagulation at the same site,
causes further platelet activation
 Upon activation, platelets change shape and in the
presence of fibrinogen, aggregate to form the hemostatic
plug
2. Secondary
 Small molecules (clotting factors), cause strands of blood-
borne materials called fibrin, together with WBCs, RBCs and
platelets to stick together and seal the inside of the wound.
 Formation of a fibrin mesh that binds to the platelet aggregate,
forming a more stable hemostatic plug or thrombus.
3. Fibrinolysis
 Eventually, the cut blood vessel heals and blood clot dissolves
after a few days.
 Partial or complete dissolution of the hemostatic plug or
thrombus by plasmin.

Trans # 10 Group # 29: Barbers, Belo, Buhat, Chan, De Guzman Trans Head: Caballar 09152258097 1 of 11
Figure 2. Blood Clot Formation within Blood Vessels (source: Lecturer’s PPT)

II. PLATELET

A. DEFINITION Figure 3. Role of Platelets in Hemostasis (source: Lecturer’s PPT)

 Non-nucleated cells present in the blood (Marks) C. PLATELET PLUG FORMATION

 Precursor: Megakaryocyte
 Extensive actin myosin system  Resting platelets are smooth and disc shaped.
 Platelet activation in response to endothelial injury causes  Activated platelets have an irregular shape with many protruding
Calcium dependent changes in the contractile elements pseudopodia
 Contains 3 types of granules:
 Electron-dense granules
 α-granules
 Lysosomal granule
 Promote the coagulation cascade involving plasma factors to
form a blood clot

Thrombopoiesis

Figure 4. Resting Platelet (1st) and Activated Platelet (2nd) (source: Dr.
Carlos’s PPT)
Adhesion
 Thrombopoietin (TPO)
 Platelet-subendothelial interaction: platelets initially adhere to the
 binds to platelet membrane receptor called mpl (CD1 10)
sites of blood vessel injury
 Produced in liver, kidney and spleen
 Influences all stages of megakaryocyte production and platelet  Collagen, subendothelial matrix-bound vWF, and other matrix
development regulation components are exposed.
 During Thombocytosis (↑ platelet count) → TPO binds to  Binding to collagen by GPIa (integrin α2β1 or VLA-2)
platelets → less TPO in the plasma → reduced stimulation of  Plate shape: Flat disc  spherical
megakaryocyte precursors  Spherical cell extrudes long pseudopods  promote platelet-
 During Thrombocytopenia (↓ platelet count) → there is more platelet interactions
TPO in the circulation → greater stimulation of megakaryocyte  Binding of subendothelial vWF by GPlb
precursors → ↑ platelet production  Changes in the platelet membrane: expose GPllb/llla (integrin
all -β3) binding sites to fibrinogen and vWF
B. FUNCTION
Activation/Secretion
 Adhesive and cohesive functions lead to the formation of a  After initial adherence  some platelets release contents of their
hemostatic plug dense granules and a-granules
 Aggregate on damaged endothelium and exposed collagen,  ADP (potent platelet activator) released from platelets and
released contents of alpha and dense granules damaged rbc binds to platelet membrane receptors -> further
 Form mechanical plugs at the site of vessel injury (Mark’s) unmasking of GPII/IIIa binding sites
 Secrete regulators of the clotting process and vascular repair  Aggregation of platelets can’t take place without promoting
(Mark’s) platelet-platelet contact and adherence
 Binding of fibrinogen to activated platelets is necessary for
Table 1. Overview of 3 Phases of Platelet Plug Formation. aggregation
Function Chracteristics  Platelet activation is inhibited by Prostacyclin (PGI2)
Adhesion Platelets roll and cling Reversible seals
to non-platelet endothelial gaps von Aggregation
surfaces willebrand factor (VWF)  Initial adherence of platelets sets off a series of reactions
necessary (platelet activation) -> more platelets are recruited and
Aggregation Platelets adhere to Irreversible platelet aggregated at the site of injury
each other plugs form requires  Occurs when platelet encounters:
fibrinogen  Released thromboxane A2
Secretion Platelets Discharge Irreversible occurs  Platelet aggregation at the site of vascular injury
contents of their during aggregation  Released ADP
granules essential to coagulation  Act on ADP receptors on platelet membrane

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 2 of

11
  Promote platelet to platelet aggregation, and release of  Both pathways converge to form FXa, which converts
more ADP and thromboxane A2 Prothrombin (FII) to Thrombin (FIIa) - only if FII is bound
* i.e. Positive feedback to Ca2+ and phospholipid membrane in the presence of FVa
 Thrombin  Thrombin therefore cleaves the inactive Fibrinogen to form
 A potent platelet granule release inducer soluble plasma Fibrin monomers (initiation of Fibrin clot
 Exposes platelet factor 3 (a negatively charged formation)
phospholipid)  The initial Fibrin clot is weak, thus converted into a more
PF3 provides a surface on which vitamin K dependent stable, insoluble fibrin clot via action of FXIIIa
factors and Ca2+ bind  This is significant because it provides more pathways in which
PF3 = platelet phospholipid we can produce/activate Factor X
 Very important because of its role in  Ultimately leading to the formation of blood clots
Enhancing platelet adhesion  Important in preventing blood ―leaking‖ from ruptured vessels that
Activating platelets may lead to shock and death
Activating factor 5, 8, 11

B. NOMENCLATURE OF BLOOD CLOTTING FACTORS

 The numbers indicate the order in which the factors have

been discovered and bear no relationship to the order in
which they act

Table 2. Nomenclature of Coagulation Factors (lifted directly from

Harper’s Illustrated Biochemistry, 30th ed.)
Factor Common Name
I Fibrinogen Note that these factors
II Prothrombin are usually referred to
III Tissue Factor by their common
names
Figure 5. Overview of Platelet plug Formation Process (source: Lecturer’s IV Ca2+ Ca2+ is usually not
PPT) referred to as a
coagulation factor
III. COAGULATION CASCADE V Proaccelerin, labile factor,
accelerator (Ac-) globulin
A. OVERVIEW OF COAGULATION CASCADE VIIa Proconvertin, serum
prothrombin conversion
accelerator (SPCA),
cothromboplastin
VIII Antihemophilic factor A,
antihemophilic globulin
(AHG)
IX Antihemophilic factor B,
Christmas factor, plasma
thromboplastin component
(PTC)
X Stuart-Prower Factor
XI Plasma thromboplastin
antecedent
XII Hageman factor
XIII Fibrin stabilizing factor
(FSF), fibrinoligase
a
There is no factor VI (discovered to be activated factor V)

 These protein coagulation factors are present in the plasma as

Figure 6. Overview of the Coagulation Cascade (source: Lecturer’s PPT)
 Blood coagulation represents a series of sequential interactions
of proteins leading to the repair of the vascular system following
injury through formation of blood clots.
 Coagulation is the simultaneous occurrence of two events
(2020C trans):
 Reaction of plasma proteins with subendothelium
 Platelets adhering to subendothelial layer
 There are 2 pathways working together: Intrinsic and Extrinsic
Pathways
 Both result in the formation of Fibrin
BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS
zymogens (proproteins) or precursors; generally serine
proteases (enzymes) and are activated by cleavage of the
polypeptide chain at one or more sites (Madarcos notes)
 The zymogens activated by cleavage cleave the next zymogen in
the cascades
 Through this sequential activation, a great acceleration and
amplification of the response is achieved
 The letter ―a‖ indicates an activated zymogen
 These plasma glycoproteins are mostly synthesized in the liver
Coagulation Factors
 Multi-domain proteins sharing conserved domains
 Generally classified into 5 types (Harper’s):
1. Zymogens of serine-dependent proteases that are
activated during the process of coagulation
2. Cofactors
3. Fibrinogen
3 of
11
 4. Transglutaminase that covalently crosslinks fibrin and  In vivo, polymers of phosphates such as extracellular DNA,
stabilizes the fibrin clot RNA, and polyphosphate (macromolecules available only
5. Regulatory and other proteins following cell damage) may serve as this negatively charged
activating surface
Regulatory Proteins  In vitro, Kaolin, a highly negatively charged hydrated
 Proteins S and C are part of the blood coagulation cascade and aluminum silicate, can be used as an initiator.
are regulatory proteins (Madarcos notes)  Leads to activation of Factor X; involves Factors XII, XI, IX, VIII,
 Pathways of regulatory proteins such as Protein C provides vital and X, as well as prekallikrein, high-molecular-weight (HMW)
biological activities in which the fluidity of circulation is kininogen, Ca2+, and cell-surface exposed phosphatidylserine,
maintained, thrombosis is prevented, and the integrity of the resulting in the production of Factor Xa (Harper’s)
vasculature in response to injury is protected.
 These proteins ensure a hemostatic balance and confined 1. When the blood vessel ruptures, inactive protein Factor
hemostatic response at the site of injury through various XII is exposed to the collagen of the matrix, causing its
mechanisms and pathways. activation.
2. Activated Factor XII (Factor XIIa) is a serine protease
Table 3. Coagulation Factors Classified into Five Types ((lifted directly and it goes on to activate another enzyme called Factor
from Harper’s Illustrated Biochemistry, 30th ed.) XI.
Zymogens of Serine Proteases 3. Activated Factor XI then activates Factor IX, which
Factor XII Binds to negatively charged surface, eg, activates Factor X.
kaolin, glass; activated by high-molecular-
weight kininogen and kallikrein
Factor XI Activated by Factor XIIa
Factor IX Activated by Factor XIa and Factor VIIa
Factor VII Activated by Factor VIIa, Factor Xa, and
thrombin
Factor X Activated on the surface of activated
platelets by tenase complex (Ca2+, Factors
VIIIa and IXa) and by the extrinsic tenase
complex Ca2+, tissue factor VIIa
Prothrombin Activated on the surface of activated
Factor II platelets by prothrombinase complex (Ca2+,
Factors Va and Xa) to form thrombin
[Factors II, VII, IX, and X are Gla-containing
zymogens] (Gla=gamma-carboxyglutamate)
Cofactors
Factor VIII Activated by thrombin; Factor VIIIa is a
cofactor in the activation of Factor X by
Factor IXa
Factor V Activated by thrombin; Factor Va is a
cofactor in the activation of prothrombin by
Factor Xa
Figure 7. Pathways of Blood Coagulation (source: Lecturer’s PPT)
Tissue Factor A glycoprotein located in the subendothelium
(Factor III) and expressed on activated monocytes to
act as a cofactor for Factor VIIa
Fibrinogen
Factor I Cleaved by thrombin to form Fibrin clot
Thiol-Dependent Transglutaminase
Factor XIII Activated by thrombin; stabilizes Fibrin clot
by covalent cross-linking
Regulatory and Other Proteins
Protein C Activated to activated Protein C (APC) by
thrombin bound to thrombomodulin; then
degrades Factors VIIIa and Va
Protein S Acts as a cofactor of Protein C; both proteins
contain Gla (gamma-carboxyglutamate)
residues
Thrombomodulin Protein on the surface of endothelial cells;
binds thrombin, which then activates Protein
C

C. PATHWAYS OF BLOOD COAGULATION

Intrinsic Pathway (Contact Factor Pathway)

 Inside the blood vessel
 Slow-acting
 Can be activated by negatively charged surfaces in vitro, for
example glass (Harper’s)
Figure 8. Simple Illustration of Intrinsic and Extrinsic Pathways converging
 Initiated by “contact” in which prekallikrein, HMW kininogen, into the Final/Common Pathway
factor XII, and Factor XI are exposed to a negatively charged (source: http://flylib.com/books/en/2.159.1.18/1/)
surface. (Harper’s)
BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 4 of
11
Extrinsic Pathway (Tissue Factor Pathway)
 Outside the blood vessel
 Quick-acting
 Calcium-dependent pathway
 Formation of fibrin clot in response to tissue injury (Harper’s)
 Leads to the activation of Factor X; involves tissue factor, Factors
VII and X, and Ca2+ resulting in the production of Factor Xa
 Initiated at the site of tissue injury, with the exposure of TF
(Harper’s)
1. When the blood vessel ruptures, it exposes a membrane
glycoprotein on the damaged cell called Tissue Factor
(TF) or Factor III.
2. TF binds to the active form of protein Factor VII, forming
a dimer protein complex (TF-7). This complex is a serine
protease; meaning it goes on to activate other enzymes
by cleaving them at specific AAs on their AA sequence
3. TF-7 complex can now activate protein Factor IX
4. TF-7 complex can also activate protein Factor X
Note: this activation of Factor X by activated Factor IX is
an amplification process of the Factor X’s formed. Factor
X is what goes on to form the blood clots.
Final/Common Pathway (Converging)
 Factor X = link between the intrinsic and extrinsic pathways
 The formation of Factor Xa is the major site where the intrinsic
and extrinsic pathways converge
1. When Factor X is activated by either pathway, it
activates Prothrombin (Factor II) to Thrombin (Factor
IIa) in the presence of other members of
Prothrombinase Complex (Platelet phospholipids, Ca2+,
Factor Va, plus Xa); this activation occurs on the surface
of activated platelets containing PS and PI
2. Thrombin (Factor IIa) converts Fibrinogen (Factor I) to
a Fibrin monomer, which eventually aggregates to form a
Fibrin clot with trapped platelets, red cells and other
components, hence white or red thrombus (see Fig. 9)
3. To strengthen the initial weak Fibrin clot, the polymerized
Fibrin monomers are cross-linked in the presence of
Factor XIIIa to form a stable, insoluble clot with
increased resistance to proteolytic and mechanical
damage (see Fig. 10)
4. The activation of Factor X into Factor Xa is the common
point between the two pathways.
Figure 9. Fibrin Clot with Trapped RBC (source: Lecturer’s PPT)
BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS
Figure 10. Cross Linking of Fibrin Molecules by Factor XIIIa (source:
Lecturer’s PPT)
D. COFACTORS
Calcium
 Some clotting enzymes in the blood (factors II, VII, IX, and
X) require calcium ion (Ca2+) to work enzymatically.
 Their binding to phospholipids in platelets and tissues
requires calcium ion to be chelated in the process.
 This happens at a post-translation modification site in the
protein clotting factor, where an extra glutamate is added to
the peptide.
 Calcium chelation at this site then allows the factor to bind
to phospholipid phosphate, by a bridge formed through the
calcium ion binding site.
Vitamin K
 Needed in the synthesis of several proteins that mediate both
coagulation and anticoagulation
 Cofactor in the activation of VItamin K-dependent proteins
(coagulation and regulatory factors)
 In the liver
 Vitamin K-dependent coagulation/regulatory factors require
activation in a lipid-rich environment
 Vitamin K = lipid-soluble
 Main source: large intestines, where gut bacteria synthesize
Vitamin K
 No bacteria = No Vitamin K = No Activation of Coagulation and
Regulatory Factors = Hemorrhage
Figure 11. Role of Vitamin K in Blood Coagulation (source: Lecturer’s PPT)
 Vitamin k acts on the precursors of clotting factors II, VII, IX, X
with carboxylation, they would be converted to more mature
forms with the GLA residue - makes them more receptive to
calcium
 Occurs inside the liver (see figure 12)
5 of
11
  Antithrombotic Effects
 Thrombin with the presence of thrombomodulin, Protein C,
Proteins S forms a complex – degrades factors Va and VIIIa,
activating them (protein s and c) thereby inhibiting these
processes/factors in the formation of clots

Figure 12. Vitamin K in Blood Coagulation (source: Lecturer’s PPT)

 The precursors become more mature versions → go out from the

liver as mature factors II, VII, IX, X → become bound with
calcium → bound to the membrane phospholipids of platelets
 This would promote the coagulation of blood

Figure 15. Antithrombotic Role (source: Lecturer’s PPT)

 Role of Serpins
 Antithrombin III (ATIII) - acts of factors I, V, VIII, XI, XIII
 Heparin - acts like ATIII forming the ATIII-heparin complex
 It potentiates ATIII
 Acts of factors IXa, Xa, XIa, XIIIa

Figure 13. Vitamin K- Dependent Carboxylation of Prozymogens (source:

Lecturer’s PPT)

E. REGULATION

 Prothrombotic Role
 Thrombin has a prothrombotic role, aside from activating
fibrinogen, it also acts on factors I, V, VII, VIII, XI, XIII

Figure 16. Role of Serpins (source: Lecturer’s PPT)

Thromboresistance of Vascular Epithelium

 Nonthrombogenic surface of normal endothelial lining
 ―This is the reason why we don’t readily form clots when there
is no damage to our blood vessels because the surface of the
normal blood vessel or when there is normal endothelial lining
is basically nonthrombogenic.” - Lecturer
 Contributing Properties
 Endothelial cells are highly negative (they would repel the
attachment of the platelet)
 Endothelial cells synthesize PGI2, PGE2, NO (these would go
against the formation of blood clots)
 Endothelial cells synthesize cofactors that inhibit thrombin
Figure 14. Prothrombotic Role (source: Lecturer’s PPT) (such as thrombomodulin and heparin SO4)

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 6 of

11
Inhibition of Excessive Clotting B. CLOT BUSTERS
Table 4. Comparison of some properties of Streptokinase (SK) and Tissue
Plasminogen Activator (t-PA)
Property SK t-PA
Selective for fibrin clot - +
Produces plasminemia + -
Reduces mortality + +
Causes allergic reaction + -
Causes hypotension + -
Cost per treatment Relatively low Relatively high

V. ANTICOAGULANT

Figure 17. Role of Fibrin in Regulation (source: Lecturer’s PPT) A. DEFINITION

 One of the ways to regulate clotting and clot lysis
 Thrombin acts to convert fibrinogen to fibrin, but fibrin acts to
inhibit thrombin as well
IV. FIBRINOLYSIS
A. DEFINITION
 Fibrinolysis is a process that occurs inside the body to break
down thrombus (blood clots)
 Involves the degradation of fibrin in a clot by plasmin. (Mark’s,
p.857)
 This prevents blood clots from remaining in place and growing
and allows the body to clear fragments of clots safely to avoid
risks such as strokes and damage to the heart that can be
caused by big floating clots
 Primary fibrinolysis refers to the normal breakdown of clots
 Secondary fibrinolysis is the breakdown of blood clots due to a
medical disorder, medicine, or other cause. This may cause
severe bleeding
 The process of Fibrinolysis:
 An anticoagulant is a substance used in order to inhibit blood
from coagulating both inside and outside the body.
 Some are medicines that help prevent blood clots, thus, reducing
their chances of developing serious conditions such as strokes
and heart attacks
 Blood clots can block blood vessels and stop blood from flowing
to organs such as brain, heart or lungs if formed in the wrong
place.
B. FUNCTION (ACTION OF ANTICOAGULANT)
1. Heparin
 Binds to and activates antithrombin AIII
 Activated antithrombin AIII then inhibits the activity of Va, IXa,
Xa, XIa
 Antithrombin AIII also causes thrombin inactivation, which
inhibits the formation of fibrin from fibrinogen, and also inhibits
actions of factors I, V, VII, VIII, XI and XIII
2. Warfarin
 structural analog of Vitamin K
 has two ways in interfering coagulation
 Warfarin blocks the activity of regulatory protein S and C,
which enhances proteolysis of Factors Va and VIIIa, this
leads to imbalance between physiologic anticoagulation
 As a structural analog of vitamin K, warfarin competes with
vitamin k in the γ-carboxylation of Factors II, VII, IX and X
(making it a vitamin K antagonist). Non carboxylation of
these factors leads to non-coagulation

3. Chelating Agents
 binds and chelates calcium to form calcium oxalate
 As calcium oxalate is formed, there is a decreas in availability
of calcium
 Without calcium, Factors II, VII, IX, X and XI cannot be
converted to their active forms, thus leads to non-coagulation

4. Direct Thrombin Inhibitors

 directly binds to thrombin inhibition leading to non-conversion
of factors I, V, VII ,VIII, IX, X and XIII to their active forms,
leading to no non-coagulation

C. FOUR MAJOR CLASSES

Figure 18. Process of Fibrinolysis (source: Lecturer’s PPT)
 Heparin
1. Plasminogen, a circulating serum protein and an inactive
 Warfarin
zymogen (homologous to the zymogens of the blood
 Chelating Agents – usually used for laboratory tests
clotting cascade), is cleaved by 1 or 2 activators to form
the active fibrin-degrading serine protease plasmin  Citrate
2. Plasmin then hydrolyzes peptide bonds of fibrin  Oxalate
degradation of fibrin into soluble degradation products →  EDTA (ethylenediaminetetraacetic acid)
clot dissolution  Direct Thrombin Inhibitor
 Bivaluridin - from hirudin in the saliva of leeches

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 7 of

11
 D. THREE MAJOR CLASSES OF DRUGS THAT INHIBIT  Because this receptor is absent or is not working properly,
BLOOD COAGULATION platelets do not stick to the injured blood vessel wall the way
they should and it is difficult for the normal blood clot to form.
 Heparin
 Warfarin
 Direct Thrombin Inhibitor
 Bivaluridin
 Fondaparinux
 Inhibits factor Xa, which interrupts blood coagulation
cascade and inhibits thrombin formation

VI. DISORDERS

A. HEREDITARY COAGULATION DISORDERS

Platelet Defect

Figure 20. Peripheral smear of patient with Bernard-Soulier syndrome

(BSS) showing giant platelets. (source:
http://emedicine.medscape.com/article/954877-overview)

3. Glanzmann Thrombastenia
 Glanzmann thrombasthenia is a platelet function disorder
that is caused by an abnormality in the genes for
glycoproteins IIb/IIIa.
 These genes code for a group of linked proteins normally
found on the surface of platelets, the glycoprotein IIb/IIIa
receptor (also called the fibrinogen receptor).
 Because this receptor is absent or is not working properly,
platelets do not stick to each other at the site of injury and it
is difficult for the normal blood clot to form.

Table 5. Summary of Platelet Coagulation Disorders: Platelet Defect (source:

Lecturer’s PPT)
Disorder Deficient Function Manifestation
Factor/
Receptor
von Willebrand vWF Adheres Mild bleeding
Disease platelets to
blood vessel
subendothelium
=> facilitates
platelet plug
formation
Bernard - Gp-Ib-IX Receptor that Mild bleeding
Soulier binds vWF to
Syndrome blood vessel
subendothelium
=> facilitates
platelet plug
formation
Glanzmaan’s Gp-IIB-IIIa Receptor that Severe
Thrombastenia binds fibrinogen bleeding
& vWF on
platelet
Figure 19. Platelet Defects (source: Lecturer’s PPT)
surfaces =>
facilitates
1. Von Willebrand Disease
platelet
 Von Willebrand disease (VWD) is a genetic disorder caused aggregation on
by missing or defective von Willebrand factor (VWF), a subendothelium
clotting protein.
 VWF binds factor VIII, a key clotting protein, and platelets in
blood vessel walls, which help form a platelet plug during the Types of Von Willebrand’s Disease
clotting process.  Type 1 vWD (quantitative and autosomal dominant)
 The condition is named after Finnish physician Erik von  mild reduction in vWF
Willebrand, who first described it in the 1920s.  Type 2 vWD (qualitative and autosomal dominant)
 Loss of high MW multimers
2. Bernard – Soulier Syndrome  Type 3 vWD (quantitative and autosomal recessive)
 Bernard-Soulier syndrome is a platelet function disorder  Severe reduction in vWF
caused by an abnormality in the genes for glycoprotein Ib-IX-
V.

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 8 of

11
Factor Defect Table 6. Summary of Platelet Coagulation Disorders: Factor Defect (source:
Lecturer’s PPT)
Disorder Deficient Function Manifestati
Factor/ on
Receptor
Protein C -
Inactivates Va
and VIIIa; Swelling of
Thromboembolis Protein C,
Protein S - lower
m Protein S
cofactor for extremities
activated
Protein C
Cofactor for
Mild
Haemophilia A VIII IX-tenase
bleeding
complex
Activates X;
Forms tenase Mild
Haemophilia B IX
complex with bleeding
VIII
Tissue factor
requires the
Immune ↓
presence of Mild to
Figure 21. Factor Defects (source: Lecturer’s PPT) Thrombocytope Phospholipi
phospholipid excessive
nic Purpura d, ↓
1. Thromboembolism for optimal bleeding
(ITP) Platelets
 Thromboembolism encompasses two interrelated conditions biological
that are part of the same spectrum, deep venous thrombosis activity
(DVT) and pulmonary embolism (PE). Cofactor of X-
Factor V Leiden Mild
 Deep Vein Thrombosis (DVT) occurs when a blood clot V Prothrombina
Thrombophilia bleeding
forms in a deep vein, usually in the leg. se complex
 Pulmonary Embolism (PE) then happens when the clot
breaks off and travels from the leg up to the lungs. B. PHARMACOLOGICAL INTERVENTION IN BLEEDING
 Protein C and Protein S act to control the formation of clot. A
defect in their production could lead to increased risk of
Thromboembolism.

2. Haemophilia A & B
 Hemophilia A, also called factor VIII (FVIII) deficiency or
classic hemophilia, is a genetic disorder caused by missing
or defective factor VIII, a clotting protein.
 Hemophilia B, also called factor IX (FIX) deficiency or
Christmas disease, is a genetic disorder caused by missing
or defective factor IX, a clotting protein.
 People with hemophilia often, bleed longer than other
people. Bleeds can occur internally, into joints and muscles,
or externally, from minor cuts, dental procedures or trauma.
 How frequently a person bleeds and the severity of those
bleeds depends on how much FVIII or FIX is in the plasma.

3. Immune Thrombocytopenic Purpura (ITP)

 Immune thrombocytopenic purpura (ITP) is a clinical
syndrome in which a decreased number of circulating
platelets (thrombocytopenia) manifests as a bleeding
tendency, easy bruising (purpura), or extravasation of blood
from capillaries into skin and mucous membranes
(petechiae).
4. Factor V Leiden Thrombophilia
 Factor V Leiden is a mutation of clotting factor V. This
mutation can increase your chance of developing abnormal
blood clots (thrombophilia), usually in your veins.
 A molecule called activated protein C (APC) prevents blood
clots from growing too large by inactivating factor V. In
people with the factor V Leiden mutation, APC is unable to
inactivate factor V normally.
 As a result, the clotting process continues longer than usual,
increasing the chance of developing abnormal blood clots.
 People with Factor V Leiden Thrombophilia have an
increased risk for venous thromboembolism (VTE).
Figure 22. Aspirin as an Anti-Platelet Aggregator (Source: Lecturer’s PPT)
 The antithrombotic action of aspirin (acetylsalicylic acid) is due to
inhibition of platelet function by acetylation of the platelet
cyclooxygenase (COX) at the functionally important amino acid
serine529
 This prevents the access of the substrate (arachidonic aid) to the
catalytic site of the enzyme at tyrosine385 and results in an
irreversible inhibition of platelet-dependent thromboxane
formation
 Aspirin is an approximately 150- to 200-fold more potent inhibitor
of the (constitutive) isoform of the platelet enzyme (COX-1) than
the (inducible) isoform (COX-2)
 Inhibition of COX-1 by aspirin will also reduce the amount of
precursors for vascular prostacyclin synthesis, provided, for
example, from adhering platelets

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 9 of

11
  Bleeding Time.
 It involves making a patient bleed then timing how long it takes for
them to stop bleeding.
 affected by platelet function, certain vascular disorders and von
Willebrand Disease—not by other coagulation factors
 ↑ during thrombocytopenia and hypofibrogenemia

Figure 23. Clopidogrel as an Anticoagulant (source: Lecturer’s PPT)

 Clopidogrel is an antiplatelet drug that reduces the expression of

the glycoprotein IIb/IIIa, the fibrinogen receptor on the platelet
surface Figure 24. Blood Smear indicating thrombocytosis
 Clopidogrel efficiently reduces ADP-induced platelet aggregation
and prolongs bleeding time B. BLOOD CLOTTING COAGULATION FACTORS ASSAY

 Partial Thromboplastin Time (PTT) or Activated Partial

Thromboplastin Time (aPTT)
 Prothrombin Time (PT)

Table 7. Summary of Blood Coagulation Tests (source: Lecturer’s PPT)

Coagulation Function Normal Pathology
Factor Assay Value
Platelet Count Quantifies 150,000 - ↓ in idiopathic
amount of 400,000 / thrombocytopenic
platelets mm3 purpura
Bleeding Time Qualitative 1-3 ↓ in
test of minutes thrombocytopenia,
platelet DIC, severe liver
function disease,
leukemia, bone
marrow failure
Partial Measures: PTT = 60 - Prolonged in
Thromboplastin - Intrinsic 70 seconds acquired or
Figure 24. Abciximab, tirofiban and eptifibatide as Anticoagulants (source: Time (PTT) or Pathway (XII, congenital def. Of
Lecturer’s PPT)
Activated XI, IX & VIII) coagulation
Partial & Common aPTT = 30 factors, Vit. K
 Abciximab, tirofiban and eptifibatide differ in chemical
Thromboplastin Pathway (X, - 40 deficiency, von
structure, binding site and pharmacokinetics
Time (aPTT) V, II & I) seconds Willerbrandt
 Abciximab is the humanized chimeric Fab fragment of a - Response Disease,
monoclonal mouse antibody; Tirofiban is a nonpeptide tyrosine to heparin leukemia,
derivative that blocks arginine-glycine-aspartic binding sites of therapy hemophilia and
GP IIb/IIIa; The cyclic heptapeptide eptifibatide blocks the lysine- heparin
glycine-aspartic binding site administration
 Abciximab, tirofiban and eptifibatide blocks the Glycoprotein (GP)
IIb/IIIa which leads to inhibition of platelet aggregation Prothrombin Measures: 11- 12.5 Prolonged in liver
Time (PT) - Extrinsic seconds disease of Vit. K
VII. BLOOD COAGULATION TEST Pathway (III, def.
VII) &
A. PLATELET ASSAYS Common
Pathway (X,
 Platelet Count V, II & I)
 Normal rnage: 150,000-400,000/mL - The
 Thrombocytopenia (<150,000/mL) effectiveness
 Excessive bleeding associated with trauma or surgery if of oral
less than 50,000/mL anticoagulant
 Thrombocytosis (high platelet count) like Warfarin
 Caused by bone marrow diseases (thromocythemia)

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 10 of

11
Table 8. Summary of Coagulation Disorders and Laboratory findings 4. Identification:
What is the normal range for platelets (platelet count)?

5. Which coagulation factor links the intrinsic and extrinsic

pathways together, forming the final common pathway?
a. Factor X
b. The X Factor UK
c. Charlie XCX
d. All of the above

6. Blood vessel wall injury initiates which of the following?

a. Common Pathway
b. Fibrinolytic Pathway
c. Extrinsic Pathway
Table 9. Differential Diagnosis of Coagulation disorders d. Intrinsic Pathway

7. Activated Protein C acts by

a. Precipitating calcium
b. Binding calcium
c. Activating plasmin
d. Inactivating VIIIa

8. All of the following conditions cause an increased

(prolonged) thrombin time except
a. Heparin therapy
b. Hypofibrinogenemia
c. Increased fibrin-degradation products
d. Decreased prothrombin

9. Which ONE of the following statements about

fibrinolysis (thrombolysis) is FALSE?
a. Fibrinolysis requires the formation of plasmin from
plasminogen.
b. Fibrinolysis is regulated by kallikreins, which are
plasminogen inactivators.
c. Tissue Plasminogen Activator (t-PA) is catalytically
inactive until it binds fibrin.
d. Fibrinolysis is important for wound healing and
recanalization of thrombosed vessels.

Thrombin Time (TT) 10. The final common pathway of coagulation involves:
source: Hatton, Chris (2008). Hematology (Lecture Notes). a. The degradation of a transglutaminase (Factor XIII).
 indicates an abnormality in the conversion of fibrinogen to fibrin
b. Formation of the prothrombinase complex.
 Normal values for thrombin time are 12 to 14 seconds.
c. Formation of prothrombin.
 the time it takes for a clot to form in the plasma of a blood
d. Formation of fibrinogen.
sample containing anticoagulant, after an excess of thrombin
Aswers: a, c, F, 150,000-400,000/mm3, a , d. d, b , b
has been added
 If the time it takes for the plasma to clot is prolonged, a
quantitative (fibrinogen deficiency) or qualitative (dysfunctional REFERENCES
fibrinogen) defect is present
Biochemistry, Lipincott’s Illustrated Reviews, Champe, P.C.,
Harvey, R.A., Ferrier, D.R., 4th ed.
Harper’s Illustrated Biochemistry, Murray, R.K., Granner, D.K.,
REVIEW QUESTIONS Rodwell, V.W., 30th ed.
Madarcos Notes: WBC and Coagulation, Madarcos, F.B., 2013
1. What enzyme is required for fibrinolysis? Mark’s Basic Medical Biochemistry: A Clinical Approach,
a. Plasmin Lieberman, ML., Marks, AD., 4th ed.
b. Warfarin Medical Biochemistry, Baynes, J.W., Dominiczak, M.H., 4th ed.
c. Thrombin Stavenuiter, F., Bouwens, E. A. M., & Mosnier, L. O. (2013). Down-
d. None of the above regulation of the clotting cascade by the protein C
pathway. Hematology Education / ... Congress of the
2. Which anticoagulant chelates and binds with Calcium to European Hematology Association. European Hematology
form Calcium Oxalate? Association. Congress. Education Program, 7(1), 365–
a. Warfarin 374.
b. Heparin Textbook of Biochemistry, Devlin, Thomas M., 7th ed.
c. Citrate Lecturer’s PPT and reording
d. Bivaluridin 2020 Trans
2019 Trans
3. T or F: AK Lectures (Youtube): Blood Clotting Cascade
The surface of a normal blood vessel is thrombogenic.

BIOCHEMISTRY HEMOSTASIS AND THROMBOSIS 11 of

11