HEMOSTATIC PHYSIOLOGY

Mansyur Arif
Dept. of Clinical Pathology
Faculty of Medicine,Hasanuddin
University, 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 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. α Granules store a variety of proteins that
are secreted by stimulated PLT. These
includes PF4, β-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 adjcent 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 Path- Half-life

way (hours)
I Fibrinogen C 90-120
II Prothrombin C 48-120
III Tissue factor I Not available
V Proaccelerin C 12-24
VII Proconvertin E 2-6
VIII Antihemophilic factor I 10-12
IX Christmas factor I 18-30
X Stuart - Prower factor I,E,C 24-60
XI Plasma thromboplastin antecedent I 45-80
XII Hageman factor I 40-70
XIII Fibrin - stabilizing factor I 72-200
HMW kininogen Fitzgerald factor I 150
Prekallikrein Fletcher factor I 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 pro-
thrombinase (converts prothrombin
 thrombin). (Fig 1).
 Prothrombin
TF
VIIa

(Ca 2+)
Xa
X
V
Lipid (TF)
(Ca 2+) 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 Prothrombin
PK
XII XIIa
HMWK

XI

XIa IXa
IX VIII
PF3
(Ca 2+)
Xa
X V
Lipid (PF3)
Test : aPTT
(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 activa-
ting 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 genera-
ted, 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 Xa
V Va Va V
PF3 TF

Thrombin

Fig.3. Autocatalytic action of thrombin. (TF= tissue fct ; PF3 = platelet fct 3)
 Prothrombin
Intrinsic system Extrinsic system

XIa TF
VIIa

IXa (Ca2+)
IX 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, parapro-
teinemias, uremia)
- heparin and abnormal fibrinogen (dysfibrinoge-
nemia) 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 pro-
longed 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 throm-
bin 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% monochloro-
acetic acid.
 XIII
Thrombin

XIIIa
Fibrin polymer Fibrin polymer
(hydrogen bonded) (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 activati-
on 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 extrin-
sically 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 counter-
balancing natural inhibitors
• In the coagulation system, antithrom-
bin 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 TF
Ca 2+ Ca 2+ Ca 2+
PL

X Xa + V
Ca 2+
PL
Prothrombin Thrombin

Fibrinogen Fibrin

XIII XIIIa 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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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 Willebrand’s 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 Finding

Hemophilia A vWF disease
Bleeding time Normal Prolonged
VIII : C Decreased Decreased
VIII : Ag Normal Decreased
VIII : RCof 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 patient’s 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
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.
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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