Hemostasis

Learning Outcomes

• Describe the hemostatic response

• Describe the events in hemostasis
• Explain the structure and functions of platelets
• Describe the process of activation of platelets
• Discuss the secondary hemostasis process
• Describe the intrinsic and extrinsic coagulation mechanisms
• Explain the different anti-clotting systems
Hemostasis
• The process of blood clotting and then the subsequent dissolution of the clot, following repair of the injured
tissue
Hemostasis
• Normal - physiological process

• Maintain blood in fluid, clot free state

• Rapid, localized haemostatic plug

• At the site of vessel injury

• The cellular elements of hemostasis -
• Vascular intima, extravascular tissue factor (TF) bearing cells, and platelets
• Intimal cells and their environment play a premier role in hemostasis
• The plasma components -
• The coagulation and fibrinolytic proteins and their inhibitors
Haemostatic Response
• Primary Phase (Primary Hemostasis) – Blood vessel and platelets

• Secondary Phase (Secondary Hemostasis) – Coagulation Factors

Primary hemostasis Secondary hemostasis

Activated by desquamation and Activated by large injuries to blood

small injuries to blood vessels vessels and surrounding tissues

Involves vascular intima and Involves platelets and coagulation

platelets system

Rapid, short-lived response Delayed, long-term response

Procoagulant substances exposed Tissue factor exposed on cell

or released by damaged or membranes
activated endothelial cells
Blood Coagulation

Hemostasis
Hemostasis is divided into three stages:
1. Vascular constriction
2. Formation of the platelet plug
3. Blood coagulation
Injury ruptures vessel. Damaged endothelial cells
release chemical signals
Vasoconstriction.
Smooth muscle contracts to constrict vessel
lumen
Platelet plug formation.
Platelets are activated by cytokines and by contact
with collagen. They become sticky and attract more
platelets. Platelet plug forms
Coagulation.
Fibrinogen is converted into fibrin, which forms a
clot with platelets and RBCs. Bleeding stops
Vasoconstriction
• Vessel spasm is initiated by endothelial
injury
• Only for small blood vessel
• Caused by local and humoral mechanisms
• Reflex contraction of smooth muscle of small
blood vessels
• Neural reflexes and thromboxane A2 (TXA2),
and serotonin contribute to vasoconstriction
• The most powerful vasoconstrictor
endothelin is released from endothelial cells
Platelets
Platelet Factors:
• From two specific types of granules (α- and δ-granules)
• - granules -Fibrinogen, von Willebrand factor (vWF), fibronectin, factors V
and VIII, PDGF
• δ-granules (dense granules) - ADP and ATP, ionized calcium, histamine,
serotonin, and epinephrine
• Other platelet factors - Glycoproteins, phospholipids, Actin, Myosin,
Thrombasthenin, Thromboxane A2
Platelet production is controlled by
thrombopoietin (TP)
Causes proliferation and maturation of
Platelet Properties: megakaryocytes
TP from - liver, kidney, smooth muscle,
▪ Adhesion – due to thromboxane A2, ADP, & von Willebrand factor and bone marrow
▪ Aggregation – activated platelets become sticky due to ADP and
thromboxane A2
Functions of Platelets
▪ Agglutination – clumping of platelets to form platelet plug • Hemostasis –Vasoconstriction and Platelet
Plug
• Blood Coagulation – Phospholipids
• For Clot Retraction – Contractile Proteins
 Platelet Plug Formation
• Platelets are attracted to a damaged vessel wall and activated
• Shape changes from smooth disks to spiny spheres, exposing
glycoprotein receptors on their surfaces
• von Willebrand factor (vWF), leaks into the injured tissue from the
plasma
• vWF is circulates in the blood as a carrier protein for coagulation
factor VIII
• Platelet receptor binds to vWF at the injury site, linking the platelet to
exposed collagen fibers

• Platelet aggregation occurs soon after adhesion

• ADP mediates platelet aggregation
• ADP also facilitates the release of ADP from other activated platelets
• TXA2 – which is another important stimulus for aggregation
• The combined actions of ADP and TXA2 → expansion of the platelet
aggregate
• A primary hemostatic plug will be formed
 PLATELET PLUG FORMATION
Platelets will not adhere to intact endothelium. Damage triggers platelet plug formation where collagen has been exposed .

Exposed collagen binds

and activates platelets.

Lumen of
blood vessel
Release of platelet factors

Prevents
platelet Factors attract more platelets.
Intact endothelium adhesion
releases prostacyclin
and nitric oxide (NO).
Platelets aggregate into
platelet plug.

Smooth Collagen Exposed collagen ECF

muscle cells subendothelial in damaged blood
layer vessel wall
Platelet Aggregation

• cAMP controls the concentration of free

calcium ions in the platelet which are
important in the processes which cause
adhesion and aggregation

• High levels of cAMP lead to low free calcium

ion concentrations and prevent aggregation
and adhesion
Platelet Aggregation
Platelets Endothelium
Thromboxane A 2 Prostacyclin (PGI2)

cAMP cAMP
cytosolic Ca 2+ cytosolic Ca 2+
++
AGGREGATION

Factors That Prevent or Inhibit Platelet Activation

Flowing blood
Prostacyclin (PGI2)
Nitric oxide (NO)
Endothelial cell CD39 (ATP diphosphohydrolase; ecto-ADPase)
Platelet refractoriness
Leukocyte–platelet interactions
Inhibitors of thrombin generation and thrombin action
 Glycoprotein (GPIIb/IIIa) receptors on the
platelet membrane bind fibrinogen and link
platelets together

Defective platelet plug formation causes

bleeding in people who
Platelet are deficient in platelets or vWF

Aggregation

Platelet aggregation inhibitor drugs (aspirin, clopidogrel ,ticlopidine)

- to prevent platelet aggregation and clot formation - in myocardial
infarction, stroke, peripheral artery disease
Blood Clotting
• Two pathways - Extrinsic and Intrinsic
• Initiated by two distinct mechanisms
• Converge on a Common Pathway
• Intrinsic pathway - activated when blood comes into contact with a negatively
charged surface
• The chain reaction occurs mainly at the membrane of activated platelets

• Extrinsic pathway - activated when blood comes in contact with material from
damaged cell membranes
• The reactions occur mainly at a tissue factor that is membrane bound
• Clot formation in response to tissue injury is the result of the extrinsic pathway
Blood Clotting Factors
Factor Trivial Name (s) Pathway
Prekallikrein
(PK)
Fletcher factor Intrinsic

High molecular
weight
kininogen
contact activation cofactor Intrinsic
(HMWK)
I Fibrinogen Both
II Prothrombin Both
III Tissue Factor Extrinsic
IV Calcium Both
V Proaccelerin, labile factor Both
VII Proconvertin, serum prothrombin conversion accelerator (SPCA) Extrinsic
VIII Antihemophiliac factor A Intrinsic
IX Christmas Factor, antihemophilic factor Intrinsic
X Stuart-Prower Factor Both
XI Plasma thromboplastin antecedent (PTA) Intrinsic
XII Hageman Factor Intrinsic
XIII Protransglutaminase, fibrin stabilizing factor (FSF), fibrinoligase Both
Regulatory Proteins

Regulatory/Other Proteins Activities

Associated with subendothelial connective tissue; serves as a bridge between
von Willebrand factor
platelet glycoprotein GPIb/IX and collagen
Activated to protein Ca by thrombin bound to thrombomodulin; then
Protein C
degrades factors VIIIa and Va

Protein S Acts as a cofactor of protein C

Protein on the surface of endothelial cells; binds thrombin, which then

Thrombomodulin
activates protein C
Most important coagulation inhibitor, controls activities of thrombin, and
Antithrombin III
factors IXa, Xa, XIa and XIIa
Clotting Factors
Vit K Dependent
Contact Factors Fibrinogen Group
Factors

XII, XI, Pre- Fibrinogen, V,

Kallikrein,HMWK II, VII, IX, X
VIII, XIII

Stable in blood Require Vit K for React with

synthesis thrombin

All consumed
Do not need Ca 2+ Need Ca2+ for
during
activation
coagulation

Not consumed in
Increases in
coagulation
pregnancy
[Except II]
Intrinsic Pathway

• A cascade of protease reactions initiated by factors that are all present within blood
• Activated when plasma comes in contact with constituents of sub endothelial
tissues
• This results in the exposure of;
- Collagen and Damaged platelets (release of phospholipids)
• The initial triggering event - the activation of FXII
• Factor XI, Pre Kallikrein and HMWK are also involved (XI, XII, HMWK, PK – contact
group)
• Autoactivation of FXII and PK occurs - whenever blood contacts an artificial surface
Intrinsic Pathway
• Factor XIIa (together with HMWK) proteolytically
cleaves factor XI to factor XIa

• Factor XIa proteolytically cleaves factor IX

• Factor IXa (and factors Xa, and thrombin)

proteolytically cleave factor VIII to VIIIa

• Factors IXa and VIIIa together with Ca2+ and

negatively charged phospholipids form a
trimolecular complex → tenase

• Tenase converts factor X to factor Xa

Intrinsic Pathway
• “Waterfall” sequence/Enzyme
cascade
• Thrombin activation leading to fibrin
clot
• Thrombin-induced ‘amplification’
and ‘propagation’
• Contact factors -
prekallikrein,HMWK, XII,XI
• Serine proteases (Factor IX and X)
and co-factors (Factor VIII, Factor V)
 Extrinsic Pathway
• Begins with activation of tissue factor
• Tissue factor (tissue thromboplastin, or factor III) – Integral membrane protein expressed in the non-vascular
cells
• Tissue factor is a receptor for factor VII
• When an injury to the endothelium allows factor VII to come into contact with tissue factor - the tissue factor
activates factor VII to VIIa
• Tissue factor, factor VIIa, and Ca2+ form a trimolecular complex, which proteolytically cleaves the factor X to
Xa
• Exposed TF forms a complex with activated factor VII, which activates factors IX and X

Layers of the endothelium showing tissue factor located in the

adventitial layer and vWF and collagen in the intima
Extrinsic Pathway

• Pathway is initiated at the site of injury

in response to the release of TF (factor
III) - tissue factor pathway
• Tissue factor is a cofactor in the factor
VIIa-catalyzed activation of factor X
• Activated factor Xa is the site at which
the intrinsic and extrinsic coagulation
cascades converge
 Final Common Pathway
• Factor Xa from either the intrinsic or extrinsic pathway -
first protease of the common pathway
• Factor V is activated to Va (thrombin increases this
conversion)
• Factors Xa and Va, together with Ca2+ and phospholipids,
form a trimolecular complex called prothrombinase
(Prothrombin activator)
• Prothrombinase and Ca+2 acts on prothrombin to form
thrombin
• Thrombin will catalyze the proteolysis of fibrinogen to
fibrin monomers (soluble fibrin)
• Fibrin monomers spontaneously polymerize to form fibrin
polymers (stable fibrin) (XIIIa helps in this conversion)
Thrombin
• Thrombin has widespread effects on multiple areas of coagulation
• Thrombin can catalyze the formation of new thrombin from prothrombin and can also catalyze the formation
of the cofactors Va and VIIIa
• Paracrine actions of thrombin
• causes endothelial cells to release nitric oxide, PGI2, ADP, vWF, and tissue plasminogen activator
• Thrombin also activates platelets to release platelet factors
• Procoagulant -
• Thrombin cleaves FXI to FXIa and FV to FVa on the platelet surface
• Also cleaves vWF off of FVIII to release FVIII and then activates FVIII to FVIIIa
• Anticoagulant -
• Protein C & Protein S with Thrombomodulin
Interaction of Coagulation Pathways
• Intrinsic pathway and extrinsic pathway are strongly
interconnected to form a network
• Trimolecular complex of [tissue factor + factor VIIa + Ca2+]
of the extrinsic pathway activates factors IX and XI of the
intrinsic pathway
• Factors IXa and Xa of the intrinsic pathway can activate
factor VII of the extrinsic pathway
• Thrombin catalyzes the formation of the cofactors Va and
VIIIa

• Clinical evidence suggests that coagulation depends largely

on the extrinsic pathway

• Inflammation can trigger peripheral blood monocytes and The in vivo pathway begins with tissue factor–factor
endothelial cells to express tissue factor, increasing the risk VIIa complex activating factor X and also factor IX.
of coagulation Factor XI is activated by thrombin

• During sepsis, the tissue factor produced by circulating

monocytes initiates intravascular thrombosis
 Clot & Clot Retraction
• Clot
• A semisolid mass composed of both platelets and fibrin
• Entrapped in the mesh of fibrin → erythrocytes, leukocytes and serum
• Thrombus is an intravascular clot
• Clot Retraction
• Normally occurs within 20- 60 min after a clot
• The process squeezes serum from the clot
• Joins the edges of the broken vessel
• Factors for clot retraction - actin and myosin from platelets
• The fibrin strands of the clot are pulled toward the platelets
 Anti - Clotting System
• Limits clot formation and dissolves clot
• Natural anti-coagulant mechanisms
• Endothelial cells are the main source of the agents that help maintain normal blood fluidity
• Defect in anticlotting mechanisms – high incidence of thrombosis [hyper coagulability]

Blood is normally in a liquid state inside blood vessels:

• It does not come into contact with subendothelial surface, that activate coagulation
pathway
• It does not contact tissue factors that activate coagulation
• Thrombolytic pathways keep the coagulation pathways in check
• Plasma contains proteins that can be converted to proteases that digest fibrin and
lyse clots
 Anticoagulant Factors from Endothelium
• Tissue factor pathway inhibitor (TFPI)
• TFPI is a plasma protein - binds to the trimolecular complex [TF + VIIa + Ca2+] in the extrinsic
pathway - blocks the protease activity of factor VIIa
• Thrombomodulin
• A glycosaminoglycan product of endothelial cells
• Forms a complex with thrombin, thereby removing thrombin from the circulation and
inhibiting coagulation
• Also binds protein C
• Protein C
• Protein C binds to the thrombomodulin portion of the thrombin-thrombomodulin complex
• Thrombin activates protein C
• Activated protein C with its cofactor protein S→ inactivates Va and VIIIa → Inhibits coagulation
 THROMBIN
Endothelium Receptor
THROMBOMODULIN

Patients with Protein C and/or Protein S deficiencies

have a thrombotic tendency.

Patients also may acquire deficiencies of Protein C

and Protein S with liver disease and disseminated
intravascular coagulation (DIC)

PROTEIN C ACTIVATED PROTEIN C

Factor Xa
Produced by endothelium &
liver PROTEIN S
Vitamin K dependent
Produced by endothelium & liver
Vitamin K dependent
INACTIVATES INHIBITORS Acts as a cofactor to Protein C
OF TISSUE PLASMINOGEN
ACTIVATOR [t-PA]
FACTOR Va & VIIIa INACTIVATED
 Anti-thrombin Mechanism
• Antithrombin III – in plasma and endothelium

• Inactivates thrombin

• Inhibit the activities of factors IXa, Xa, XIa

• Binds to activated factors rendering them inactive

• Action of AT-III is enhanced and accelerated by the presence of

heparin

• Heparin binding to Antithrombin III – activation of AT III

• Patients with decreased AT-III levels → an increased risk of

thromboembolism
Fibrinolytic System PLASMINOGEN ACTIVATORS
Intrinsic activators: Extrinsic Activators:
Tissue Plasminogen Activator [t-PA]
Kallikrein Urokinase Type PA [u-PA]
XIIa
Liver, plasma, and vascular
endothelium are the major
+ Streptokinase sources of physiologic
+ activators

PLASMINOGEN PLASMIN

+ +
FIBRINOGEN FIBRIN

DEGREDATION PRODUCTS
FIBRIN DEGRADATION PRODUCTS
• The process begins with the conversion of plasminogen to plasmin
• Plasminogen - mainly made by the liver
• t-PA, from endothelial cells
• u-PA, is present in plasma
• Plasmin is a serine protease - breaks down fibrin and fibrinogen
 Anticoagulant Therapy
• Heparin
• Heparin is naturally formed and released in small amounts by mast cells in connective tissue surrounding
capillaries

• Inhibition of blood clotting – rapid action

• Binds to antithrombin III, causing a conformational change that increases the ability of AT III to inactivate
thrombin, factor Xa, and other clotting factors

• Doesn’t cross the membranes of the gastrointestinal tract

• Warfarin

• Oral anticoagulant – readily absorbed

• Decreases prothrombin and other procoagulation factors

• Takes a couple days for its action

• Inhibits production of vitamin K dependent factors

Pharmacological Anti-platelet agents
• Aspirin

• Antiplatelet therapy

• Irreversible inhibition of the platelet

enzyme cyclooxygenase, which is
needed for platelet aggregation

• Affects last for about a week