HEMOSTATIC &

COAGULATION PHYSIOLOGY

Regulation of hemostatic
mechanism involves complex
interaction between vessels walls,
blood cell elements and a variety of
plasma proteins.

Blood clotting results from :

1. Interaction between endothelial cells and
platelets the primary hemo-static plug.
2. The coagulation phase thrombin is
generated & fibrin develops
3. Formation of peptide bonds
stabilization of the fibrin network.

Fibrinolysis is the process of

enzymatic degradation of fibrin clots
whereby : coagulation activity is
limited to the area surrounding
vessels wall injury and patency of
vessels is maintained or restored

PLATELET STRUCTURE
Major Structure features:
- A typical cell membrane.
- Circumferential mcrotubular system
- Dense tubular system
- Various granules
- Externally communicating open
canalicular system.

Platelet Membrane
Bilipid membrane and membrane protein.
Bilipid membrane around the platelet
contains several important glycoproteins
that function as surface receptors.
Bilipid membrane is also the site of
complex coagulation activities of the
platelet.

Bilipid membrane (cont.)

1. Glycoprotein Ib (GP Ib). MW of about
140 kD. It serves as the binding site for
vWf.
2. Glycoprotein IIb-IIIa (GP IIb-IIIa).
A prominent Ca-dependent membrane
protein complex that function as a
fibrinogen receptor.

Microtubules and Microfilaments

1. Microtubules are composed of
tubulin and participate in cytoskeletal
support and in contraction of the
stimulated cell.
2. Microfilaments contain actin and
participate in platelet pseudopod
formation.

Dense Tubular System

- Electron dense material.
- Selectively binds divalent cations and
serves as the platelet Ca reservoir.
- Site of PLT cyclooxygenase and of
prostaglandin synthesis.

Granules
1. Dense granules contain high concentrations
of ADP and Ca as well as serotonin. These
substances
are
released
upon
PLT
stimulation,enhance PLT aggregation.
2. Granules store a variety of proteins that
are secreted by stimulated PLT.
These
includes PF4, -tromboglobulin, PDGF,
Fibrinoen, F V, vWf and various glycoproteins
important to adhesion (thrombospondin &
fibronectin)

Canaliculi
Open canalicular system is a complex
network
of
surface
membrane
invaginations that look like vacuoles.
Increase the PLT surface area.
The contents of PLT granules are
released through this system

Platelet Physiology

When a blood vessels is injured:

- Subendothelial tissue is exposed.
- PLT adhere to Subendothelial tissue.
- Adherence mediated by vWf form a bridge
between subendothelial tissue and GP Ib.
- Thrombin stimulates membrane
phospholipids to release archidonic acid .

Platelet Physiology
AA is converted to cyclic endoperoxides
and TxA2.
Stimulate granules and dense bodies.
High concentration locally thrombin, TxA2
and ADP will change GP IIb-IIIa becomes
receptor for fibrinogen to forms a bond
between adjacent PLT creating a hemostatic
plug.

Phospholipid
Phospholipase
Arachidonic acid
Cyclo-oxygenase
(aspirin inhibits)
PGG2
Peroxidase
PGH2
Thromboxane synthetase

Prostacyclin synthetase

(platelets)

(endothelium)

TxA2

PGI2
H2O

TxB2

6-keto PGIa

Fig. 8. Arachidonic acid metabolism in pletelets endothelium.

Endothelium Contribution
Metabolize AA to Prostacyclin (PGI2).
PGI2 has major contribution as
antithrombotic in intact endothelium.
Low dose aspirin completely block TxA2
production.

Coagulation phase :
1. Conversion of fibrinogen (soluble plasma
protein) into insoluble fibrin affected by
highly specific enzymatic action of
thrombin.
Thrombin must be generated from
zymogen, prothrombin, by a series of
reactions between serine proteases, co
factors & lipid moieties.

2. Coagulation factors may be group as

follows : contact factors, thrombin sensitive factors, & vit K- dependent
factors.
Classically, the generation of thrombin
is described as occuring through the
extrinsic or intrinsic systems.
Table 1 summarizes the features of the
coagulation factors.

Table 1. Plasma coagulation factors

Factor

Alternative name

Pathway

Half-life
(hours)

I
II
III
V
VII
VIII
IX
X
XI
XII
XIII
HMW kininogen
Prekallikrein

Fibrinogen
Prothrombin
Tissue factor
Proaccelerin
Proconvertin
Antihemophilic factor
Christmas factor
Stuart - Prower factor
Plasma thromboplastin antecedent
Hageman factor
Fibrin - stabilizing factor
Fitzgerald factor
Fletcher factor

C
C
I
C
E
I
I
I,E,C
I
I
I
I
I

90-120
48-120
Not available
12-24
2-6
10-12
18-30
24-60
45-80
40-70
72-200
150
48-52

Coagulation systems :
1. The extrinsic systems : triggered by TF/
tissue factor (complete thromboplastin).
- TF + VIIa + Ca activates F X (F Xa)
- F Xa + V + Lipid (TF) extrinsic prothrombinase (converts prothrombin
thrombin). (Fig 1).

Prothrombin
TF
VIIa
Xa
V
Lipid (TF)
(Ca 2+)

(Ca 2+)
X

Test : PT
(Quick)

Thrombin
Fig.1. Generation of thrombin via the extrinsic system. (TF = tissue fct ;
PT = prothrombin ; = prothrombin complex ; = F X activating
complex).

2. The intrinsic system :

a. Contains all the elements necessary for
clotting.
b. Instead of tissue thromboplastin, the lipid
moiety in this system is PF3.
c. The contact fcts (F XII, XI, prekallikrein/PK,
High Molecular-Weight Kininogen/HMWK) are
activated by exposure to negatively charged
glass surfaces & other substances
(ellagic
acid,uric acid crystals,skin,collagen
&
antibody complexes)

d. - F XIIa in the presence of PK and

HMWK activates F XI.

- F XIa activates IX, which in a complex
with VIII, lipid (PF3) and Calcium
activates F X.
- F Xa, V and lipid (PF3) comprise
intrinsic prothrombinase. (fig. 2).

Contact factors
XII

PK
HMWK

Prothrombin

XIIa
XI

IX

XIa

IXa
VIII
PF3

(Ca 2+)
X
Test : aPTT

Xa
V
Lipid (PF3)

(Ca 2+)

Thrombin
Fig.2.

Generation of thrombin via the intrinsic system. (PK = prekallikrein; HMWK/

high molecular weight kininogen ; PF3 = platelet factor 3 ; aPTT = activated
partial thrombplastin time ;
= prothrombinase complex ;
= F X activating complex.

e. - Screening test : aPTT screens for all

the coagulation factors except F VII.

- intrinsic & extrinsic pathways
converge at
the F X and V level.
- A coagulation factor deficiency (or
inhibitor) at this level results in
abnormal screening test for both
system.

f. F VIII & V are cofactors for F IXa & Xa.

When initial traces of thrombin are generated, F VIII & V are activated (VIIIa & Va).
Larger amounts of thrombin results in
destruction of these factors. (Fig.3)
g. Interlinkage between the intrinsic & extrinsic
systems occurs at several levels. The most
important of these is the ability of TF and
factor VIIa to activate F IX. (Fig.4)

Prothrombin
Intrinsic system

Extrinsic system

IXa
VIII
VIIIa
PF3

Xa
V

Xa

Va
PF3

Va

V
TF

Thrombin
Fig.3. Autocatalytic action of thrombin. (TF= tissue fct ; PF3 = platelet fct 3)

Prothrombin
Intrinsic system
Extrinsic system
TF
VIIa

XIa
(Ca2+)
IX

IXa
VIII
PF3

(Ca2+)
X

Xa

Thrombin
Fig. 4. Linkage between extrinsic and intrinsic systems. Several interaction
occur at various levels of the two systems. Primary among these is the
ability of TF and F VIIa and F IX.

3. Fibrin generation
When thrombin acts on the fibrinogen
molecu-le, two pairs of tiny fibrinopeptides (A
& B) are cleaved off, yielding activated fibrin
mono-mer units. The monomers polymerize
to form a loose, unstable fibrin clot, which
can be converted to a stable fibrin clot. (Fig.5)

Thrombin
Fibrinogen
Fibrin
Fibrin Polymer
monomer (hydrogen bonded)
Fig. 5. Fibrin generation

a.

Screening test : Thrombin time (TT)

TT is prolonged due to :
- fibrinogen concentration is very low (<80 mg/dl)
- interfere with polymerization of fibrin monomer
(fibrin (ogen) degradation product/FDP, paraproteinemias, uremia)
- heparin and abnormal fibrinogen (dysfibrinogenemia) are present.

b. Additional test
In the reptile time test, the snake venom
employed selectively cleaves fibrinopeptide A
from the fibrinogen molecule. Clotting will
proceed even though fibrinopeptide B
remains intact.
This test can be valuable because it is prolonged in the same circumstances as the TT
except that is not prolonged by the presence
of heparin.

Fibrin stabilization
Final stage of coagulation
F XIII, a transaminase, is activated by thrombin and converts the hydrogen-bonded fibrin
strands into more stable, covalent peptide
bonds. (Fig.6)
Screening test :
Deficiency of F XIIIa results in clots that
dissolve in 5M urea or 1% monochloroacetic acid.

XIII
Thrombin
XIIIa
Fibrin polymer
(hydrogen bonded)

Fibrin polymer
(peptide bonded)

Fig. 6. Fibrin stabilization. The initially formed clot of polymers of

fibrin monomer is stabilized by thrombin activated F XIII.
F XIIIa converts the fibrin strands into covalently bonded,
stable fibrin.

Fibrinolysis
Deposition of fibrin is associated with activation of fibinolysis
Fibrin is a substrate for the proteolytic action
of plasmin.
Plasmin is normally present in its inactive,
zymogen form (plasminogen) in blood, urine
and other body fluids.
Plaminogen may be activated intrinsically by
the contact system of coagulation or extrinsically by TPA/tissue plasminogen activator.

 Exogenous activation may be induced

therapeutically by administration of
urokinase or streptokinase. (Fig.7)
Screening Test :
TT may be prolonged due to presence
of FDP (may show elevation).
.

Physiologic inhibitors
Procoagulant & fibrinolytic activities are
homeostatically regulated by counterbalancing natural inhibitors
In the coagulation system, antithrombin III inhibits not only thrombin but
other serine protease as well (F IXa,
Xa, XIa, XIIa).

 Protein C along with its cofactor, protein

S, degrades F VIIIa and Va.
Plasmin is neutralized primarily by
2 - antiplasmin.

Plasminogen
Intrinsic system

Extrinsic system

Contact
XII

XIIa
HMWK
Exogenous

Prekallikrein

Kallikrein

TPA

Urokinase
Streptokinase

Plasmin
Fig.7.

The fibrinolytic system. Plasmin, the active fibrin(ogen)olytic enzyme,

is generated by activation of plasminogen as shown.
= plasminogen activator.

INTRINSIC SYSTEM
HMWK
XII
XII a
Kallikrein
XI

XIa

EXTRINSIC SYSTEM
VII

IX

IXa + VIII
Ca 2+
PL

Ca 2+

TF
Ca 2+

Xa + V
Ca 2+
PL

Prothrombin

Thrombin
Fibrinogen

XIII

XIIIa

Fibrin

Stable fibrin clot

Ca 2+

II. TESTS OF COAGULATION SYSTEM

A. Screening Test

1. Partial thromboplastin time (PTT) and

activated partial thromboplastin time (aPTT)
2. Prothrombin time (PT)
3. Quantitative fibrinogen
4. Thrombin time (TT)
5. Screening test for factor XIII

B. Spesific factor assays

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