CLOT FORMATION AND LYSIS FunctionConsequences 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 SYSTEM ComponentFunction Platelets and von Willebrand factorFormation of primary hemostatic plug Coagulation cascadeFormation of fibrin clot (stabilizes and strengthens hemostatic plug) Vessel wall/endotheliumEndothelium antithrombotic; vasoconstriction limits bleeding from injured site Fibrinolytic systemControlled clot lysis allows wound healing

PLATELETS

PLATELETS ,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 COMPONENTS Organelles –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 RECEPTORS Thrombin ADP receptor (several forms) Thromboxane Prostacyclin P-selectin Thrombopoietin (modulates TPO concentration in blood) ß2-adrenergic receptor Adenosine receptor Serotonin receptor

PLATELET 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 INHIBITORS Activators (partial list) Collagen (in subendothelium) ADP (from rbc, activated platelets) Thromboxane A 2 (from activated platelets) Thrombin Epinephrine Inhibitors Nitric oxide (from endothelium) Prostacyclin (Prostaglandin I 2 - from endothelium) Aspirin (blocks thromboxane synthesis) Clopidogrel (Plavix ® - blocks ADP receptor)

Platelet activation and inhibition by prostaglandins

PLATELET ADHESION TO AREA OF VESSEL INJURY

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

PLATELET AGGREGATION

VON 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 FACTOR Single very large molecules visualized by electron microscopy Electrophoresis showing range of multimer sizes

VWF UNFOLDS UNDER SHEAR STRESS The faster the blood flow, the stickier it gets

Von Willebrand factor

REGULATION OF VWF SIZE BY ADAMTS-13

COMPONENTS OF THE PLATELET RESPONSE

FIBRIN 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 coagulation ProteinSize (kD) Concentration (mg/dl)Type of proteinFunction (after activation) Fibrinogen (factor I)340300StructuralPolymerizes to form clot Prothrombin (factor II)72.510VKZ* Activates fibrinogen, V, VIII, XIII, protein C, platelets, XI Factor V3502HelperSupports activation of II by Xa Factor VII500.1VKZActivates IX and X Factor VIII3500.1HelperSupports activation of X by IXa Factor IX571VKZActivates X Factor X591VKZActivates II Factor XI1600.5ZymogenActivates IX Factor XIII3203ZymogenCrosslinks fibrin, other proteins *VKZ = vitamin K-dependent zymogen

TISSUE FACTOR LARGE VESSELMONOCYTE MONOCYTE + ENDOTOXIN SMALL VESSEL Am J Pathol 1989; 134: 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 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 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 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” 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 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 ENDOTHELIUM Sequesters tissue factor from blood Prostaglandin I 2 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 mechanism T T T (antithrombin) (substrate)

THE ANTITHROMBIN SYSTEM

E C TM THE PROTEIN C SYSTEM A negative feedback loop that degrades factors Va, VIIIa IIa P S APC Va Vi VIIIa VIIIi P C

TF VIIa X TF VIIa Xa TFPI TF VIIa Xa TFPI Tissue factor pathway inhibitor

FIBRINOLYSIS 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)

FIBRINOLYSIS

FIBRINOLYSIS Intact fibrin clotFibrin clot exposed to plasmin