CLOT FORMATION AND LYSISCLOT FORMATION AND LYSIS

Function Consequences of dysfunction

Rapid formation of mechanically sound clot at areas of injury

Bleeding

Prevent clotting in areas removed from injury

Thrombosis

Controlled clot lysis and replacement by connective tissue

Bleeding (if lysis too fast)

Thrombosis (if too slow)

Poor healing

COMPONENTS OF THE HEMOSTATIC SYSTEMCOMPONENTS OF THE HEMOSTATIC SYSTEM

Component Function

Platelets and von Willebrand factor Formation of primary hemostatic plug

Coagulation cascade Formation of fibrin clot (stabilizes and strengthens hemostatic plug)

Vessel wall/endothelium Endothelium antithrombotic; vasoconstriction limits bleeding from injured site

Fibrinolytic system Controlled clot lysis allows wound healing

PLATELETSPLATELETS

PLATELETSPLATELETS

• 150-400,000/μl blood

• Lifespan in blood 7-10 days

• 30% sequestered in spleen (more if spleen enlarged)

• Megakaryocyte development under influence of thrombopoietin and other growth factors

PLATELET ORGANELLES AND MEMBRANE COMPONENTSPLATELET ORGANELLES AND MEMBRANE COMPONENTSOrganelles

– Mitochondria– Glycogen granules (energy source)– Lysosomes– Dense granules (serotonin, Ca++, ATP, ADP)– Alpha granules (various plasma proteins, growth factors etc)

Membrane glycoproteins– GP Ib/IX/V: von Willebrand factor receptor (mediates platelet

adhesion); 50K copies/platelet– GP IIb/IIIa (αIIbß3 integrin): fibrinogen receptor (mediates

platelet aggregation; cryptic in resting platelets); 50-80K copies/platelet

Membrane phospholipids (inner leaflet)– Anionic/procoagulant– Arachidonic acid– “Flippase” keeps procoagulant molecules on inner leaflet –

they are exposed with platelet activation

SOME IMPORTANT PLATELET MEMBRANE RECEPTORSSOME IMPORTANT PLATELET MEMBRANE RECEPTORS

• Thrombin• ADP receptor (several forms)• Thromboxane• Prostacyclin• P-selectin• Thrombopoietin (modulates TPO concentration in blood)• ß2-adrenergic receptor• Adenosine receptor• Serotonin receptor

PLATELET FUNCTIONPLATELET FUNCTION

• Maintain vessel integrity– Red cells leak from uninjured vessels if platelets absent

(petechiae)– This effect mediated by various factors released by

platelets, e.g., VEGF, angiopoietin, EGF• Help plug holes in damaged vessels

– Adhesion (von Willebrand factor, GPIb)– Shape change– Activation – Release of granule contents– Aggregation (fibrinogen, GP IIb-IIIa)– Provide large membrane surface for thrombin

generation, fibrin clot formation– Clot retraction

PLATELET ACTIVATORS AND INHIBITORSPLATELET ACTIVATORS AND INHIBITORS

Activators (partial list)• Collagen (in subendothelium)• ADP (from rbc, activated platelets)• Thromboxane A2 (from activated platelets)• Thrombin• Epinephrine

Inhibitors• Nitric oxide (from endothelium)• Prostacyclin (Prostaglandin I2 - from endothelium)• Aspirin (blocks thromboxane synthesis)• Clopidogrel (Plavix® - blocks ADP receptor)

Platelet activation and inhibition by prostaglandinsPlatelet activation and inhibition by prostaglandins

PLATELET ADHESION TOPLATELET ADHESION TOAREA OF VESSEL INJURYAREA OF VESSEL INJURY

PLATELET SPREADINGPLATELET SPREADING

Patel et al, Blood 2003;101:929-36

PLATELET AGGREGATIONPLATELET AGGREGATION

VON WILLEBRAND FACTORVON WILLEBRAND FACTOR

• Large multimeric protein made by endothelial cells (and megakaryocytes)

• Released into blood and subendothelium– Secretion stimulated by stress, exercise, epinephrine, thrombin and

vasopressin analog DDAVP

• Mediates platelet adhesion via GP Ib receptor• Largest multimers most effective (very large multimers

broken down after secretion)• Immobilization and unfolding of VWF (collagen binding,

shear stress) increases adhesiveness• Deficiency of VWF causes bleeding tendency (von

Willebrand disease)• VWF in blood binds factor VIII and protects it from

inactivation

VON WILLEBRAND FACTORVON WILLEBRAND FACTOR

Single very large molecules visualized by electron microscopy

Electrophoresis showing range of

multimer sizes

VWF UNFOLDS UNDER SHEAR STRESSVWF UNFOLDS UNDER SHEAR STRESSThe faster the blood flow, the stickier it gets

Von Willebrand factorVon Willebrand factor

REGULATION OF VWF SIZE BY ADAMTS-13

COMPONENTS OF THE PLATELET RESPONSE

FIBRIN CLOT FORMATIONFIBRIN CLOT FORMATION

• Tissue factor (in subendothelium and other tissue) triggers enzymatic cascade that results in formation of thrombin– Platelet membrane supports and catalyzes

these enzymatic reactions

• Thrombin converts fibrinogen to fibrin

• Fibrin polymerizes, polymers are crosslinked to form stable clot

Plasma proteins involved in coagulationPlasma proteins involved in coagulation

Protein Size (kD)Concentration (mg/dl) Type of protein Function (after activation)

Fibrinogen (factor I) 340 300 Structural Polymerizes to form clot

Prothrombin (factor II) 72.5 10 VKZ*Activates fibrinogen, V, VIII, XIII, protein C, platelets, XI

Factor V 350 2 Helper Supports activation of II by Xa

Factor VII 50 0.1 VKZ Activates IX and X

Factor VIII 350 0.1 Helper Supports activation of X by IXa

Factor IX 57 1 VKZ Activates X

Factor X 59 1 VKZ Activates II

Factor XI 160 0.5 Zymogen Activates IX

Factor XIII 320 3 Zymogen Crosslinks fibrin, other proteins

*VKZ = vitamin K-dependent zymogen

TISSUE FACTORTISSUE FACTOR

LARGE VESSEL MONOCYTEMONOCYTE

+ ENDOTOXIN

SMALL VESSEL

Am J Pathol 1989; 134:1087-97

Tissue factor is a ubiquitous, membrane-associated lipoprotein that is expressed by most cells. It is not normally expressed by endothelial cells or leukocytes, although expression can be induced in these cells by inflammatory cytokines (above, right). There is high expression in the adventita of blood vessels (above, left), brain, skin and mucosal tissue (the “hemostatic envelope”).

Generation of thrombin. Each step in the coagulation cascade requires an enzyme (in this case factor Xa), a proenzyme substrate (in this case prothrombin), a “helper” protein (here it is factor Va), a phospholipid surface (eg, a platelet membrane) and calcium. The enzyme and its substrate proenzyme contain calcium-binding regions that are vitamin K-dependent.

a

γ-carboxy glutamyl residues (vitamin K-dependent)

Blood 2013;122:2773

VITAMIN KVITAMIN K

• Fat-soluble vitamin present in many foods– Some also made by gut bacteria

• Needed to create calcium-binding sites on several clotting factors (II, VII, IX, X) and anticoagulant proteins (protein C, S)

• Deficiency can cause bleeding tendency

• Warfarin (Coumadin®) is an antagonist of vitamin K, used as an anticoagulant

Initiation of coagulationInitiation of coagulation

• Tissue factor exposed to blood, binds VIIa– Small amounts of VIIa in normal plasma

• TF/VIIa activates X and IX• IXa/VIIIa activate X• Xa/Va activate II to form thrombin

– Initial activation of V probably by Xa

• TF/VIIa complex quickly inhibited by Tissue Factor Pathway Inhibitor (TFPI)

• Amount of thrombin generated as a result of these reactions may be insufficient for clot formation

Propagation (amplification) of coagulationPropagation (amplification) of coagulation

• IIa activates XI, VIII, V (positive feedback)

• Xa and IIa activate VII

• XIa generates more IXa

• IXa/VIIIa generate more Xa

• Xa/Va generate more IIa

• IIa converts fibrinogen to fibrin (and XIII to XIIIa)

• Fibrin crosslinked by XIIIa

The “Contact System”The “Contact System”• When plasma is exposed to a negatively charged surface,

this proteolytic pathway is activated• 3 components: high molecular weight kininogen,

prekallikrein, and factor XII• The end product of contact activation is factor XIIa, a

serine protease that can activate factor XI• This system is responsible for the fact that blood clots

when exposed to glass and other foreign surfaces• Polyphosphate released from platelets supports activation• Complete deficiency of any of the contact factors does not

cause bleeding, suggesting that this pathway has little if any role in physiologic coagulation– System may contribute to regulation of fibrinolysis, angiogenesis,

and inflammatory response

Blood 2005;106:2944

Thrombin

Fibrinogen

Fibrin monomer

Fibrin polymer

FIBRIN FORMATIONFIBRIN FORMATION

Platelets (red)Tissue factor (green)Fibrin (blue)Platelets + tissue factor (yellow)Tissue factor + fibrin (turquoise)Platelets + fibrin (magenta)Platelets + fibrin + tissue factor (white)

Clot formation in a mouse following vascular injury

Celi et al, J Thrombos Haemost 2003;1:60

Fibrinogen

Tissue factor-VIIa

IXa-VIIIa

Xa-Va

Initiation

Propagation

Thrombin

Fibrin

AT3

APC

TFPI

Regulation by anticoagulants (black boxes). TFPI- tissue factor pathway inhibitor, AT3- antithrombin, APC- activated protein C. Red arrows indicate inhibitory activity.

ANTITHROMBOTIC PROPERTIES OF ANTITHROMBOTIC PROPERTIES OF ENDOTHELIUMENDOTHELIUM

• Sequesters tissue factor from blood

• Prostaglandin I2 inhibits platelet aggregation

• Heparan sulfate catalyzes inhibition of thrombin by antithrombin

• Thrombomodulin catalyzes activation of protein C by thrombin

• Nitric oxide is a vasodilator and inhibits platelet aggregation

• ADPase inhibits platelet aggregation

Antithrombin mechanismAntithrombin mechanism

T

T

T

(antithrombin)

(substrate)

THE ANTITHROMBIN SYSTEMTHE ANTITHROMBIN SYSTEM

E CTM

THE PROTEIN C SYSTEMTHE PROTEIN C SYSTEMA negative feedback loop that degrades factors Va, VIIIaA negative feedback loop that degrades factors Va, VIIIa

IIa

IIa

P S

APC

VaVi

VIIIa

VIIIi

P C

TF

VIIa

X

TF

VIIa

Xa

Xa XaTFPI

TF

VIIa

Xa

TFPI

Tissue factor pathway inhibitorTissue factor pathway inhibitor

FIBRINOLYSISFIBRINOLYSIS

• Proteolysis of clot and replacement by connective tissue

• Conversion of plasminogen to plasmin by plasminogen activator starts the process

• Plasminogen activation catalyzed by fibrin• Alpha 2 antiplasmin and plasminogen activator

inhibitors regulate the process• Products of fibrinolysis can be measured in blood

(fibrin split products, D-dimer)• Plasminogen activators are useful in treatment of

some thrombotic conditions (MI, stroke, etc)• Excessive fibrinolysis can cause bleeding (eg, DIC)

FIBRINOLYSISFIBRINOLYSIS

FIBRINOLYSISFIBRINOLYSIS

Intact fibrin clot Fibrin clot exposed to plasmin