Hemostasis and Bleeding

disorders

Questions?
Why dont we normally form blood clots in
normal and uninjured blood vessel?
How and why do we form clots when our
small vessels are injured?

Normal hemostasis

Normal Hemostatic mechanisms:

Maintain blood in a fluid, clot free state
Induce formation of a hemostatic plug at the
site of vessel injury to prevent blood loss.
Normal hemostasis is regulated by :
1. Blood vessel wall (endothelium)
2. Platelets
3. Coagulation cascade
4. Fibrinolytic agents
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Lets see what happens when a small

vessel in injured or severed ?

Sequence of events in hemostasis after

vessel injury ..

Four phases .
1. Vascular phase
2. Platelet phase
3. Coagulation phase
4. Fibrinolytic phase

Vascular phase

Platelet phase

Coagulation phase

Fibrinolytic phase

Vascular phase

1. Vasoconstriction occurs immediately

after injury.
2. Factor VII is activated by tissue
thromboplastin (tissue factor)
3. Exposed collagen activates factor
XII.

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Platelet phase
Adhesion: GpIb receptors on the platelets
adhere to exposed vWF in damaged endothelial
cells.
Release reaction: release of ADP causes platelet
aggregation.
Synthesis and release of TXA2: vessels constrict
, reducing blood flow; this action further
enhances platelet aggregation
Temporary platelet plug stops bleeding:
aggregated platelets with fibrinogen attached to
their GpIIb-IIIa receptors form a plug.
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 Coagulation phase:
Thrombin produced by activation of
coagulation cascade converts fibrinogen
into insoluble fibrin to form a stable
plug.
Fibrinolytic phase:
Plasmin breaks insoluble fibrin to reestablish the blood flow

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Vascular
phase

Vessel injury with activation of coagulation cascade

Platelet adhesion to collagen
(by GpIb receptor for vW factor)

Platelet phase

Platelet release of aggregating agents (ADP)

Platelet synthesis of TXA2 (vasoconstrictor and aggregating agent)
Platelet aggregation
Formation of temporary platelet plug
= primary hemostatic plug
-Cessation of bleeding
-End of bleeding time

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Formation of temporary platelet plug

= primary hemostatic plug
-Cessation of bleeding
-End of bleeding time

Coagulation phase

Formation of stable platelet plug

(by action of thrombin on fibrinogen)

Fibrinolytic phase

Dissolution of platelet plug by plasmin

Restoration of blood flow

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Role of blood vessels in normal

hemostasis
Endothelial cells:
Modulate opposing aspects of hemostasis
Provide an
Anticoagulant function in the uninjured
state and a
Procoagulant function when injured.

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Endothelial cells: anticoagulant functions

Is due to synthesis of different substances:
1. Heparin like molecules:
Enhance antithrombin III activity.
AT III neutralizes coagulation factors.
Factors XII,XI,X,IX,II and thrombin
2. Protein C and S
Vitamin K dependent factors
Inactivate factors V & VIII
not inactivated by AT III
Also enhance fibrinolysis
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Endothelial cells: anticoagulant functions

3. Prostaglandin I2 (PGI2) : prostacyclin
Vasodilator and
inhibits platelet aggregation.
4. Tissue plasminogen activator (tPA)
Activates plasminogen to produce plasmin.
Plasmin:
degrades coagulation factors &
lyses fibrin clots
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Note: Natural anticoagulants

1. Role of AT III
Heparin stimulates AT III
Activated AT III inactivates
thrombin and factors XII,XI,IX,X and
II.
2. Role of protein C and S
Binding of thrombin to thrombomodulin on
endothelial cells activates protein C.
Activated protein C and S
inactivate factors V,VIII and enhance
fibrinolysis.
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Endothelial cells: Procoagulant functions

Procoagulants released in small vessel injury:
Thromboxane A2 (TXA2): Synthesized by platelets
Is a vasoconstrictor and platelet aggregator
PGH2 converted to TXA2 by thromboxane
synthase.
Aspirin irreversibly inhibits platelet
cyclooxygenase
Prevents formation of PGH2, the precursor
of TXA2
Tissue thromboplastin (factor III) :
Released from injured tissue
Activates factor VII in the extrinsic coagulation
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system.

Endothelial cells: Procoagulant functions

von Willebrands factor (vWF):
Synthesized by endothelial cells &
megakaryocytes.
Functions of vWF:
Platelet adhesion molecule
Binds platelets to exposed collagen
Platelets have GpIb receptors for vWF
Complexes with factor VIII coagulant
(VIII:C) in the circulation.
VIII:vWF complex prevents degradation
of factor VIII:C
Decrease in vWF secondarily decreases20
VIII:C activity*.

Role of endothelial cells

Summary
Intact endothelium serves to inhibit platelet
adhesion and blood clotting.
Injured or activated endothelium augments
local clot formation.

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Role of Platelets in normal hemostasis

Derivation:
Cytoplasmic fragmentation of megakaryocytes.
Membrane components:
Glycoprotein receptor for vWF : GpIb
Glycoprotein receptor for fibrinogen:
GpIIb:IIIa
Platelet factor 3 (PF3):
Located on the platelet membrane
Phospholipid substrate required for the
clotting sequence.
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Platelets
Structure:
Alpha granules contain:
vWF, fibrinogen
Platelet factor 4 (PF4)
heparin neutralizing factor
Dense bodies or delta granules contain
ADP: an aggregating agent,
Calcium (Ca2+) : a binding agent for vitamin K
dependent factors
Contractile elements:
Thrombosthenin
Helps in clot retraction.
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Role played by Coagulation cascade in

normal hemostasis
Main objective: to convert soluble protein
fibrinogen to insoluble fibrin.
This is brought about by interactions of
coagulation factors (soluble plasma proteins).
The reactions occur in a water fall like fashion
leading to sequential activation of coagulation
factors.
Ultimately leading to formation of thrombin.
Thrombin converts soluble fibrinogen into
insoluble fibrin.
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Coagulation factors

Total of 12 factors
Named according to order of discovery
No factor VI
The factors are proenzymes that must be
converted to active form.
An activated coagulation factor has enzymatic
activity (e.g. activated factor XII XIIa).
Some conversions occur on phospholipid surface
Some conversions require calcium.
Source: synthesized in liver ( severe liver disease
bleeding tendency).
Some factors require vitamin K dependent
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carboxylation to function.

COAGULATION FACTORS
FACTOR

NAME

Fibrinogen

II

Prothrombin

III

Tissue factor= thromboplastin

IV

calcium ions = ca++

V*

No factor VI

VII
VIII

Antihemophilic factor

IX
X

Stuart factor

XI
XII

Hageman factor

XIII

Fibrin stabilizing factor

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Coagulation cascade

Coagulation cascade divided into THREE

interacting pathways:
1. Extrinsic
2. Intrinsic and
3. Final common pathway

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XII

Intrinsic system
Collagen
HMWK

Extrinsic system
Tissue
thromboplastin

XIIa
VII

XI

VIIa

XIa

IX

IXa

VIII+IXa+PF3+Ca2+
Xa

X
V+Xa+PF3+Ca2+

Thrombin

Prothrombin

Fibrin monomer

Fibrinogen
Fibrin
monomer aggregate

Soluble fibrin
XIII

XIIIa
Thrombin

Cross linked
insoluble fibrin
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Coagulation cascade
Extrinsic pathway: factor VII
Begins with activation of factor VII
Tissue thromboplastin released from injured
tissue activates factor VII resulting in
formation of factor VIIa
VII

Tissue thromboplastin

VIIa

In the final common pathway factor VIIa

activates factor X.
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Coagulation cascade

Intrinsic pathway : factors XII,XI,IX & VIII

Begins with activation of factor XII
(Hageman factor)
Exposed subendothelial collagen and HMWK
activate factor XII to form factor XIIa
(activated Hageman factor)
Factor XIIa activates Factor XI to form
factor XIa

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Intrinsic pathway
XIa activates factor IX to form IXa
IXa complexes with factor VIII, Ca++ and
PF3 to form a four component complex
(factor IXa, factor VIII, PF3, Ca2+).
In the final common pathway, this complex
activates factor X.

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 Final common pathway: factors X,V, II

(prothrombin) and I (fibrinogen).
Begins with activation of factor X by:
Factor VIIa (extrinsic pathway)
Four component complex [(factor IXa,
factor VIII, PF3, Ca2+) intrinsic pathway].
Activated factor Xa complexes with factor V,
PF3 and Ca++ to form a complex =
Prothrombin complex
Prothrombin complex : cleaves prothrombin to
the enzyme thrombin.
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 Functions of Thrombin:
Acts on fibrinogen to produce soluble
fibrin monomers.
Activates fibrin stabilizing factor XIII
Activated fibrin stabilizing factor
XIIIa converts soluble fibrin monomers
to insoluble fibrin
by enhancing cross linking between
proteins to strengthen the fibrin
clot.

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Vitamin K dependent factors

Consist of factors II, VII, IX ,X and Proteins C
& S.
All synthesized in liver as nonfunctional
precursor proteins.
Functions of vitamin K:
Vitamin K is activated in liver by Epoxide
reductase
Activated vitamin K, carboxylates each
factor
Carboxylated factors are functional
Can bind to calcium and PF3 in the cascade
sequence.
Epoxide reductase is inhibited by Warfarin 34

Pharmacologic anticoagulants
Heparin:
Increases Antithrombin III activity.
Warfarin ( coumadin)
Inhibits vitamin K dependent
carboxylation of factors II,VII,IX and
X
Present in rat poison

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Fibrinolytic system
Breaks down the stable clot in order to restore
blood flow.
Activated by:
tPA : activates plasminogen to form the
enzyme plasmin
Functions of plasmin:
Cleaves insoluble fibrin monomers and
fibrinogen into fibrin degradation products
(FDPs).
Fragments of cross linked insoluble fibrin
monomers are called D-dimers.
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Laboratory evaluation of hemostasis

Platelet tests:
1. Platelet count
2. Bleeding time
3. Test for vWF: Ristocetin cofactor assay
Coagulation system tests:
1. Prothrombin time (PT)
2. Activated partial thromboplastin time (aPTT)
3. Thromboplastin time (TT)
4. Mixing studies
5. 5 molar urea clot solubility test
Fibrinolytic system tests:
1. FDP assay
2. D dimer assay

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Platelet tests
Platelet count:
most cost effective test
Normal count is 150,000 to 400,000
cells/uL.
A normal count does not guarantee normal
platelet function.
Bleeding time:
Evaluates platelet function upto the formation
of temporary platelet plug.
Normal range: 2-7 min
Abnormal if defect in platelet
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Number or Function

Causes of prolonged bleeding time

Cause

Nature of defect

Aspirin or NSAIDs

Platelet aggregation defect

Bernard-Soulier
syndrome
Glanzmanns disease
Renal failure
Thrombocytopenia
Von Willebrands
disease
Scurvy

( TxA2)

Platelet adhesion defect

( GpIb)

Aggregation defect ( GpIIb-IIIa)

Aggregation defect
( platelet phospholipid)

Decreased number ( platelets)

Adhesion defect (vWF)
Vascular defect (Vit C)

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 Tests for vWF:

Ristocetin cofactor assay
Evaluates vWF function
Abnormal assay
Classic vW disease (deficiency of
vWF)

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Coagulation system tests

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42

Prothrombin time
Evaluates the extrinsic & common pathway down
to the formation of a clot.
Tests factors: VII,X,V,II,I.
A deficiency of one or more of the above
factors prolongs the PT.
Normal range: 11 to 15 sec.
International normalized ratio (INR):
Standardizes the reporting of PT results in
patients taking warfarin.
Results are the same regardless of the
reagents used to perform the test.
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Prothrombin time
Uses of PT:
Follow patients who are taking warfarin
for anticoagulation.
PT elevated
Evaluating the severity of liver disease.
Elevated PT indicates severe liver
dysfunction
Confirming factor VII deficiency
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Activated partial thromboplastin time

(aPTT)

Tests the intrinsic & final common pathways

down to the formation of a clot.
Tests factors: XII,XI,IX,VIII,X,V,II,I.
Normal range: 25 to 40 sec
Uses of PTT:
Follow heparin therapy**.
Heparin enhances AT III activity
Detect factor deficiencies in intrinsic system
Elevated in hemophilia and heparin therapy.
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Fibrinolytic system tests

Fibrin (ogen) degradation product (FDP assay):
detects all breakdown products of fibrinogen
or insoluble fibrin clots by plasmin.
D dimer assay:
Only detects cross linked insoluble fibrin
monomers in a fibrin clot.
Does not detect FDPs (not cross linked)
Most specific test for evidence of degradation
of a fibrin clot (thrombus); examples:
Thrombolytic therapy for CAD.
Screening for DIC* & pulmonary emboli

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