HEMOSTATIC PHYSIOLOGY

Mansyur Arif
Dept. of Clinical Pathology
Faculty of Medicine,Hasanuddin University,
Makassar
 Hemostasis is the biochemical process
that protects the body from loss of blood
after vascular damage.

Vessel contraction and platelet aggregation

stop bleeding immediately (within second)
and trigger the coagulation cascade
 The coagulation cascade is a chain reaction
in which inactive enzymes are converted to
their active form.
The cascade ends with fibrinogen to fibrin
conversion, catalysed by activated thrombin.
In the presence of activated factor XIIIa the
fibrin is cross-linked and clotted to an
insoluble thrombus (fibrin-clot).
The bleeding is stopped.
 All factors and inhibitors are balanced
very carefully.
In the case of any imbalance or
dysfunction, serious vascular diseases
can and do appear.
Dysfunction of complex hemostasis is
one of the most common vascular
diseases, and often results in death (1
per 1000 patients).
Examples are deep-vein thrombosis
(DVT) and Pulmonary embolism (PE)
 Normal coagulation is a complex
sequence of reactions between plateles,
endothelium and coagulation factors
Primary hemostasis platelet activation
and platelet plug formation
Secondary hemostasis activation of
coagulation cascade and formation of
stable fibrin complex.
Fibrinolysis then limits the extent of
thrombosis.
 Symptoms of mucosal bleeding, such as
epistaxis, gum bleeding, hematochezia,
melena, petechiae, or easy bruising
signs of defective primary hemostasis
secondary to thrombocytopenia, pletelet
dysfuntion, or abnormalities of von
Willebrand factor (vWF)
Hemarthroses, i.m hemorrhage, bleeding
into deeper structure >> secondary
hemostasis disorder with a coag. factor
deficiency or dysfunction
 Laboratory test in the work up of
coagulation disorder
The first lab test : CBC (+peripheral
slide), PT, aPTT, Thrombin Time (TT) .
aPTT activity of intrinsic factor
PT activity of extrinsic factor
TT common pathway
Bleeding time <<, but evaluate
problems with primary hemostasis
CBC first step in evaluation of bleeding patient
thrombocytopenia, anemia, leukocytosis
or leukopenia implicate a hematologic
malignancy cause of patients
coagulopathy or thrombocytopenia.
PB evaluation platelet clumping and
WBC morphology
Coagulation Test
Prothrombin time measure of extrinsic factor
(tissue factor) and common pathway
 Prolongation of PT caused by : antagonis
vit K (warfarin ingestion), deficiency
(inadequate dietary intake), decrease in
vit-K dependent , direct thrombin inhibitors
or quantitative or qualitative disorder of
fibrinogen.
PT values varied from institution to
institution INR (International
Normalized Ratio)
Activated Partial Thromboplastin Time
(aPTT) intrinsic and common pathways.
Prolonged aPTT and associated with
increased bleeding : Fct IX, XI, VIII
Total def of F XII, prekallikrein and high
molecular-weight kininogen (HMWK)
prolonged aPTT but not increase
bleeding .
Deficiencies and inhibitors of common
pathway prolonged aPTT.
 Thrombin Time (TT)
Measures the final common step in
the coag. cascade (fibrinogen fibrin)
Prolonged in coag. disorders including
hypo- and dysfibrinogenemia, high
levels of fibrinogen degradation
product, monoclonal gammopathies or
thrombin inhibitors (lepirudin or
argatroban), thrombin antibodies,
exposure to heparin.
No Test Platelet Condition
PT APTT TT count
1 Long N N N Factor VII deficiency
Early oral anticoagulant

2. N Long N N Factor VIII, IX, XI, XII deficiency

Prekallikrein, HMWK deficiency
Von Willebrands disease
Anticoagulant

3. Long Long N N Vitamin K deficiency

Oral anticoagulant
Factor V,VII, X dan II deficiency

4. Long Long Long N Heparin

Liver disease
Fibrinogen deficiency
Hyperfibrinolysis

5. N N N Low Thrombocytopenia

6. Long Long N Low Massive transfusion

Liver disease

7. Long Long Long Low DIC

Acute liver disease

First-line tests used in investigating acute haemostatic failure

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 arachidonic 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 platelets endothelium.

 Endothelium Contribution
Metabolize AA to Prostacyclin (PGI2).
PGI2 has major contribution as
antithrombotic in intact endothelium.
Low dose aspirin completely block TxA2
production.
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 factors (F XII, XI, prekallikrein
/PK, High Molecular-Weight Kininogen/
HMWK) are activated by exposure
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.
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+
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.
 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.5)
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. 5. 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 fibrinolysis
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 extrin-
sically by TPA/tissue plasminogen activator.
Physiologic inhibitors
Procoagulant & fibrinolytic activities are
homeostatically regulated by counter-
balancing 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.
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