HEMOSTATIC

PHYSIOLOGY
Presented by:
Agus Alim Abdullah
Department of Clinical Pathology
Faculty of Medicine
University of Hasanuddin Makassar

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 microtubular 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,
Fibrinogen, 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.
Plasminogen 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

III. CONGENITAL HEMORRHAGIC DISORDER

A. Hemophilia A (factor VIII deficiency) and
Hemophilia B (factor IX deficiency)
1. Pathophysiology :
X chromosome, gene defect.
2. Clinical features :
mild, moderate and severe disease
3. Diagnosis :
a. screening tests
b. specific factor assay

4. Therapy : to raise the deficient factor

a. Pharmacologic therapy :

1. Hemophilia A : Desmopressin (dDAVP)

2. Hemophilia B : no effective drugs
b. Replacement therapy :
1. Beware of adverse effects
2. The choice of blood product is critical

c. Treatment options :
1. Fresh frozen plasma (FFP)
2. Cryoprecipitate
3. Factor concentrates
5. Complication : Arthropathy, Inhibitors,
Liver disease, HIV infection.
6. Interdisciplinary care hemophilia center
medical care, psychosocial care and
genetic counseling

B. von Willebrands disease

1. Physiology of vWF :
- HMW glycoprotein, 250 kD
- In plasma and in platelets
- as a carrier protein for coag. F VIII
- important for primary hemostasis
- mediates adhesion of platelets to the s.endot
- vWF F VIII complex : F VIII : C, F VIII : Ag,
F VIII : Cag, F VIII : RCof.

2. Clinical features : >> mucous membrane

bleeding, epistaxis, menorrhagia
3. Diagnosis :
- Combination of prolonged BT and a
decreased F VIII : C level classically
- Combination of abnormalities of the functional
measures of the vWF F VIII complex

variants of vWF (see table 2)

- Ristocetin aggregation
- Diff. of type F VIII multimer analysis :
type I, IIA, IIB, IIC, III.

Table 2. Laboratory Findings in Hemophilia A

and Severe vWF Disease
Labotatory test
Bleeding time
VIII : C
VIII : Ag
VIII : RCof

Finding
Hemophilia A
vWF disease
Normal
Prolonged
Decreased
Decreased
Normal
Decreased
Normal
Decreased

4. Therapy
a. dDVAP
b. Cryoprecipitate
c. F VIII concentrates

C. Other inherited factor deficiencies

1. F XII, prekallikrein and HMWK
2. F I, II, V, VII, X, XI and XIII

IV. ACQUIRED HEMORRHAGIC DISORDER

A. Vitamin K deficiency
1. Etiology
a. dietary deficiency
b. malabsorption
c. antibiotic therapy
d. hemorrhagic disease of the new born
2. Clinical features : severe bruising or
excessive bleeding or asymptomatic

3. Diagnosis
- severe : prolonged PT and PTT
- early or milder def. : prolonged PT
4. Therapy
- Parenteral vit. K
- FFP
B. Liver disease
1. Etiology
a. decreased synthesis of coag. factors
b. Vit. K def

c. Functionally abnormal fibrinogens

d. Disseminated Intravasc. Coagulation
(DIC) and consumption of coag. factors
2. Clinical features
- vary with the course of the patients liver
disease
- >> GIT bleeding
3. Diagnosis
- Lab. findings varies widely

4. Therapy
- trial of parenteral vit K therapy
- Replacement therapy FFP
C. Clotting factor inhibitors autoantibodies
1. Inhibitors in hemophilia
2. Inhibitors in patients without preexisting
bleeding disorders
a. Etiology : drugs, autoimmune or
lymphoproliferative disorders,
spontaneous a.coagulants

b. Diagnosis : mixing study, factor assays

c. Therapy : supportive
3. Lupus anticoagulant cardiolipin
a. Prolonged PTT, false + serologic test
b. associated with recurrent spont.abortions.

COAGULATION REGULATION
AND
HYPERCOAGULABLE STATES

Control Mechanism in Coagulation

- Soon after coagulation is initiated, several
regulatory mechanisms are activated to
inhibit coagulation.
- Uncontrolled autoamplification of
coagulation would result in generalized
thrombosis.

I. CONTROL MECHANISMS IN COAGULATION

A. Naturally occuring a.coagulants
1. Antithrombin III (AT III) acts as a serine
protease inhibitor
2. Protein C, Protein S Vit K dependent
3. Other plasma protease inhibitor : heparin
cofactor II, 2- macroglobulin.

Site of action
AT III can inhibit many serine protease
enzymes (Thrombin and factors Xa, IXa
and XIa.
Thrombins central role in coagulation
the most critical site of inactivation.

Protein C
(inactive form of Protein Ca)
Serine protease that acts on specific clotting
factors in the presence of cofactor protein S.
Protein C with the presence of thrombin and
thrombomodulin inhibits on fibrinogen and
platelets.

Sites of action
Coagulation. In the presence of phospholipid and
with protein S as cofactor will inactivates factors
Va and VIIIa.
Fibrinolisis. With protein S as cofactor, protein Ca
enhances fibrinolisis by preventing degradation of
plasminogen avtivator by its inhibitor.
Protein Ca activity is limited by protein C
inhibitor.

B. Fibrinolitic system
1. Activation :
a. Intrinsic activation : a poorly understood
mechanism of activation
b. Extrinsic Activation : Tissue plasminogen
activator (t-PA), urokinase
c. Exogeneous (therapeutic) activation :
streptokinase, urokinase and t-PA

2. Sites of action
a. Fibrinogen
b. Fibrin
II. THROMBOTIC DISORDER
A. Congenital thrombotic disorder
1. AT III deficiency
2. Protein C deficiency
3. Protein S deficiency

B. AQUIRED THROMBOTIC DISORDER

THROMBOEMBOLIC DISEASE
1. Risk factors :
a. abnormalities of blood flow
b. abnormalities of the vasculature
c. abnormalities of the coagulation system
2. Clinical manifestations :
a. Venous thrombosis
- superficial thrombosis
- deep vein thrombosis

b. Pulmonary embolism
c. Arterial thrombosis
d. Other
4. Therapy
a. Agents treatment and prevention
1. Anticoagulants :
- Heparin
- Warfarin
2. Thrombolytic agents :
- Streptokinase
- Urokinase
- t-PA

b. Treatment approaches
1. Deep vein thrombosis : >> heparin,
thrombolytic agent
2. Pulmonary embolus : heparin, thrombolytic
agent
3. Myocardial infarction : thrombolytic agent
4. Peripheral artery and catheter thrombosis :
thrombolytic agent.

III. THROMBOHEMORRHAGIC DISORDER

A. Disseminated intravascular coagulation(DIC)
1. Pathophysiology (see table 3)
a. Initiation : pathologic activation of
the coag. cascade
b. Thrombosis
c. Consumption
d. Fibrinolysis
e. Hemolysis

Table 3. Conditions that commonly precipitate DIC

Infectious condition
Gram negative septicemia
Other endotoxin-related condition
Obstetric conditions
Abruptio placentae
Amniotic fluid embolism
Retained dead fetus
Vascular conditions
Aneurysm
Giant cavernous hemangioma
Hematologic conditions
Massive hemolysis
Promyelocytic leukemia
Snake venom
Trauma

2. Clinical features varies widely

a. diffuse bleeding from multiple sites
b. thrombotic lessions
3. Diagnosis lab. findings vary with time &
circumstances
Severe DIC :
a. Thrombocytopenia
b. Prolonged PT, PTT, TT
c. Decreased fibrinogen
d. FSPs
e. Microangiopathic hemolytic anemia

4. Therapy :
a. Low grade DIC treatment may not
be necessary
b. Clinically significant bleeding
replacement of depleted coagulation
factors and cells
c. Thrombosis : heparin

B. Thrombotic thrombocytopenic purpura

(TTP) and hemolytic-uremic syndrome
(HUS)
C. Heparin thrombocytopenia

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