CHAPTER 37 part 1

NORMAL HEMOSTASIS AND

COAGULATION
Normal Haemostasis and thrombosis 1
INTRODUCTION
• Circulating blood (in fluid state)
• If injury occurs -
• Clot forms to stop bleeding
• Clot confined to injured site
• Clot then dissolves as healing takes place
• Imbalance in hemostasis – haemorrhage (bleeding) or thrombosis
(pathological clotting)
• Major systems of haemostasis: blood vessels, platelets and plasma
proteins

Normal Haemostasis and thrombosis 2

INTRODUCTION
Hemostasis definition:
“complex physiologic process that keeps circulating blood in a fluid
state and then, when an injury occurs, produces a clot to stop the
bleeding, confines the clot to the site of injury, and finally dissolves the
clot as the wound heals”.
Clot aka hemostatic plug, blood clot, or thrombus is the actual barrier
that forms to limit blood loss.

Normal Haemostasis and thrombosis 3

Interaction of three components required if haemostasis:
• Blood vessels
• Platelets
• Coagulation factors (i.e. soluble plasma proteins)

Normal Haemostasis and thrombosis 4

OVERVIEW
Interaction of vasoconstriction, platelet adhesion and aggregation, and
coagulation enzyme activation to stop bleeding.
Key cellular elements of hemostasis:
• the cells of the vascular intima,
• extravascular tissue factor (TF)–bearing cells, and
• platelets

Normal Haemostasis and thrombosis 5

OVERVIEW
Stages of haemostasis:
• Primary haemostasis – primary haemostatic plug forms
• Secondary haemostasis – secondary haemostatic plug forms
• Fibrinolysis – clot dissolved and removed.

Normal Haemostasis and thrombosis 6

Key cellular elements:
• Cells of vascular intima (eg endothelial cells)
• Extravascular tissue factor (TF)-bearing cells
• Platelets

Key plasma component:

**Coagulation protein/factors
**Fibrinolytic proteins
Inhibitors to the **
Normal Haemostasis and thrombosis 7
OVERVIEW
Primary haemostasis: (vascular intima and platelets involved)
i.e. role of blood vessels and platelets in response to a vascular injury, or to
desquamation of dying or damaged endothelial
cells.
• Blood vessels contract to seal the wound or reduce the
• blood flow (vasoconstriction).
• adhere to the site of injury,
• Platelets become activated,
• secrete the contents of their granules,
• and aggregate with other platelets to form a platelet plug.
Normal Haemostasis and thrombosis 8
OVERVIEW
Primary haemostasis:
• Primary haemostatic plug temporary (i.e. not stable) and can easily
dislodge from the vessel wall.
• Short-lived, rapid response
• The plug has to be reinforced by fibrin to prevent major bleeding in
the long term.
[Defect of primary haemostasis: collagen abnormalities,
thrombocytopenia, qualitative platelet disorders, or von Willebrand
disease]

Normal Haemostasis and thrombosis 9

OVERVIEW
Secondary haemostasis (platelets and coagulation system involved)
• Inactive proteins (in plasma) aka zymogens or proenzymes, are
activated
• In active form, they form complexes that activate other zymogens.
Thrombin is generated
• Thrombin converts fibrinogen to fibrin.
• The fibrin strands are deposited on and within the primary platelet
plug to reinforce and stabilize it.
• Clot now known as secondary haemostatic plug.

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OVERVIEW
Fibrinolysis
Final stage
Clot dissolved or gradually digested
Fibrin clot removed as wound heals

SEE TABLE 37-1 page 643

NB:IF INJURY OCCURS TO AN INTACT BLOOD VESSEL, BLOOD CLOT FORMS ON AN INTERIOR SURFACE OF THE
DAMAGED BLOOD VESSEL. THIS CONDITION IS CALLED THROMBOSIS

Normal Haemostasis and thrombosis 11

VASCULAR INTIMA IN HEMOSTASIS
Endothelial cells:
• form inner most lining of the blood vessels
• Principle elements in regulating many vascular functions
• In healthy blood vessels: endotothelial cells are nonthrombogenic and
antithrombotic, preventing inappropriate clotting
• Damaged vessels: endothelial cells become thrombogenic, i.e.
promoting the formation or a blood clot

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VASCULAR INTIMA IN HEMOSTASIS
Endothelial cells (cont) function:
Normal endothelium: platelets and coagulation proteins are inert
Physiologically, a nonreactive environment is provided by haemostatic
functions that inhibit clot formation:
Endothelial cell surface is negatively charged and repels platelets and
circulating proteins that are also negatively charged.
Biochemically, EC synthesize and secrete a wide variety of substances
which contribute to the non-reactive environment.
See box 37-1 page 643

Normal Haemostasis and thrombosis 13

 VASCULAR INTIMA FUNCTION
IN HEMOSTASIS
OF ENDOTHELIAL CELLS
COMPONENT/CHARACTERISTIC FUNCTION
Haemostatic function:
(i) Nonthrombogenic/anticoagulant function
Negatively charged Repels platelets and haemostatic proteins
Heparan sulfate Inhibits fibrin formation (cofactor for antithrombin)
Also enhances the activity of antithrombin
Thrombomodulin Inhibits thrombin formation (cofactor in activation of
protein C;
PGI2 (Prostacyclin) (synthesized in thromboxane Vasodilation; inhibits platelet aggregation
pathway ( see chapter 13)
Tissue plasminogen activator (tPA) Activate the fibrinolytic system
Nitric oxide Vasodilator; induces smooth muscle relaxation, inhibits
platelet activation
Tissue factor pathway inhibitor Controls activation of the tissue factor pathway

EC expresses endothelial protein C receptor (EPCR) which binds protein C AND works together with
thrombomodulin to catalyse the protein C pathway
Protein C = natural inhibitor and inhibits/digest FV Haemostasis
Normal and FVIII.and thrombosis 14
[SEE BOX 37-2]
VASCULAR INTIMA IN HEMOSTASIS
FUNCTION OF ENDOTHELIAL CELLS
COMPONENT/CHARACTERISTIC FUNCTION
Hemostatic function:
(ii) Thrombogenic / procoagulant function
Endothelin Vasoconstrict
VWF production and processing Carries FVIII in plasma; causes platelet adhesion to
exposed collagen
Tissue factor Released during injury and initiates fibrin formation
P selectin (CD62) Promotes platelet and leukocyte adhesion

Steps: Injury; Vasoconstriction

Collagen exposed
Secretes platelet activating factor which activates platelets.
EC secretes VWF which is needed for platelets to adhere to collagen
Exposes TF and activates the coagulation cascade through contact with FVII.
Normal Haemostasis and thrombosis 15
VASCULAR INTIMA IN HEMOSTASIS
FUNCTION OF ENDOTHELIAL CELLS
COMPONENT/CHARACTERISTIC FUNCTION
Hemostatic function:
(iii) Fibrinolytic properties
Tissue plasminogen activator Activate the fibrinolytic system by converting
plasminogen to plasmin. Plasmin digests fibrin and
restores blood flow.
Plasminogen activator inhibitor 1 (PAI-1) Inhibits plasmin generation
Thrombin-activatable fibrinolysis inhibitor (TAFI) Inhibits plasmin generation
Activated by thrombin bound to thrombomodulin

Increased PAI-1 & TAFI slows fibrinolysis and leads to increased thrombotic tendencies.

Normal Haemostasis and thrombosis 16

Overview of Hemostasis(cont) aka the haemostasic
response (ADDITIONAL INFORMATION)
PRIMARY HAEMOSTASIS/ PLATELET PLUG FORMATION:
Vascular intima and platelet
A) Vasoconstriction
 Injury (small) to vessel – immediate constriction of adjacent small arteries

and arterioles to slow blood flow

B) Platelet reactions and primary haemostatic plug formation
 Steps involved in platelet plug formation

 Step1: Injury to endothelium

 Collagen is exposed
 VWF binds to collagen (VWF is synthesized in endothelial cells and
megakaryocyte; stored in Weibel Pallade bodies and platelet α granules
released by the endothelialNormal
cells)
Haemostasis and thrombosis 17
 Role of platelets in haemostasis
• maintenance of blood vessel integrity
• Platelet–platelet interactions (primary hemostatic plug)
• Platelet–coagulation protein interactions (secondary
hemostatic plug)
• Aid in healing injured tissue

Normal Haemostasis and thrombosis 18

 Role of platelets in haemostasis
Platelet–platelet interactions (primary hemostatic plug):
Sequence of events:
Injury, Vasoconstriction
adhesion,
contraction or shape change,
secretion, and
aggregation

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Tissue injury
Vasoconstriction to reduce the blood flow
ADHESION:
• After tissue injury Collagen exposed
• (At low shear rates platelet binds directly to their collagen
receptor via GPIa/IIa
• At high shear rates von Willebrand factor (vWF) is required.
• VWF binds to collagen .
• More platelet rush to the site.
• VWF then binds to platelets via GPIb/IX/V which is present on
the platelet surface.
Bernard SOULIER syndrome:
• VWF therefore now becomes the bridge between collagen mutations in one of the genes of the
and platelets GPIb/IX complex that causes either
• VWF can also bind to platelets via GPIIb/IIIa decreased amounts of the receptor
• [GPIa/IIa has a higher affinity for collagen, but GPVI is a more complex on the platelet sur-face or
abnormal function of the complex.
effective platelet activator. The result of their interaction is
firm adhesion and platelet activation. GPVI activation induces Von Willebrand Disease:
ADP release from the dense granules (DG) and synthesis of mutations in the gene encoding VWF
thromboxane A2 (TXA2) from arachidonic acid].
Normal Haemostasis and thrombosis Source: McKenzie & Williams, 20
2015
PLATELET ACTIVATION:
• After adhesion morphologic and shape change of platelets
• GPIIb/IIIa, a receptor for fibrinogen is generated.
• Agonists bind to surface receptors on the platelet and induce a series of reactions
within platelets.
• Platelets change their shape, forming projections called pseudopods

Secretion and aggregation

• After shape change, platelets secrete their granular content. The open canalicular
system fuses with the membranes of the granules.

Aggregation
• GPIIb/IIIa binds soluble fibrinogen
• New platelets rush to the area and they are activated by coming into contact with
agonists such as ADP and TXA2.
• ADP causes platelets to swell
• TXA2 causes calcium (Ca2+) to be released and promotes platelet aggregation and
vasoconstriction.
• Fibrinogen now serves as a bridge and cross-links GPIIb/IIIa.
Diseases associated with defective aggregation:
Glanzman thrombasthaenia : lacks GPIIa/IIIb or abnormal GPIIb/IIIa Source: McKenzie & Williams, 2015
afibrinogenaemia
Glanzmann thrombastenia: GPIIb/IIIa dieficient or abnormal due to mutations in αIIIβ3
Normal Haemostasis and thrombosis 21
 Platelet Granule Contents

Platelet alpha granules Platelet dense granules

(Dense bodies)
Platelet a-Granules Platelet Dense Granules (Dense Bodies)
Large Molecules Small Molecules
β-Thromboglobulin Adenosine diphosphate (activates neighboring platelets)
Factor V Adenosine triphosphate
Factor XI Calcium
Protein S Serotonin (vasoconstrictor)
Fibrinogen
VWF
Platelet factor 4 (heparin inhibitor)
Platelet-derived growth factor

Normal Haemostasis and thrombosis 22

During activation:
ADP & Ca2+ activates phospholipase A2
Phosholipase A2 converts Membrane phospholipid to arachidonic
acid which in turn is converted into prostaglandin endoperoxides.

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DURING ACTIVATION

Membrane ADP & Ca2+ activates phospholipase A2

phospholipid Phosholipase A2 converts Membrane
Phospholipase A2 (activated phospholipid to arachidonic acid which in
by ADP and Ca2+ Ca2+) turn is converted into prostaglandin
Arachidonic acid endoperoxides
Cyclooxygenase
Prostaglandin
endoperoxides

Normal Haemostasis and thrombosis 24

 Inside the platelet The aspirin effect:
Prostaglandins
Aspirin = antiplatelet drug
which inhibits cyclooxygenase.
 Thromboxane synthetase TXA2 not produced and
therefore platelets not
 Thromboxane A2
causes functioning properly
(TXA2)
 Ca2+ released

Platelet aggregation
and vasoconstriction

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 Once primary platelet plug is formed, the platelet plug needs to be
further stabilized. If not, the primary plug can easily dislodge and
bleeding can continue.

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Factors that modulate platelet plug formation
• Proaggregation/procoagulation factors: Platelets release
ThromboxaneA2 (TXA2) which decreases blood flow and increases
platelet aggregation
• Anti-aggregation/anti-coagulation factors: (ProstaglandinI2 (PGI2 ) and
nitric oxide) are released by endothelial cells lead to an increase in
blood flow and decrease in aggregation to wash the clot away
• [NB: Procoagulation and anticoagulation are opposite forces who
work at the same time]

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• Platelet membrane is the key surface for coagulation enzyme-
cofactor-substrate complex formation.

Normal Haemostasis and thrombosis 28

Normal Haemostasis and thrombosis 29
 Platelets (Cont.)
 Other blood cells
 Erythrocytes: add bulk and integrity to the fibrin clot
 Monocytes and lymphocytes: provide surface-borne
tissue factor that may trigger coagulation
 Leukocytes in general: provide integrins and selectins
that bind adhesion molecules and help stimulate the
production of cytokines that promote the wound-
healing process

Continued
Normal Haemostasis and thrombosis 30
 References

 Keohane, E.M. Otto, C.N. Walenga, J.M. 2020. RODAK’s

Hematology. CLINICAL PRINCIPLES AND APPLICTIONS. Elsevier.

 Mckenzie, S.B. 2014. Clinical Laboratory Hematology. 2nd ed. Pearson.

 Hoffbrand, A.V. Moss, P.A.H. 2016. Essential Hematology. 7th ed. Wiley-
Blackwell.

Normal Haemostasis and thrombosis 31