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Hemostasis and thrombosis  Normal hemostasis is a consequence of a tightly regulated processed that maintain blood in a fluid state in normal

vessels  Permit rapid formation of hemostasis clot at the site of a vascular injury  Pathological counter part of hemostasis - thrombosis – involves blood clot (thrombus_ formation within intact vessels  3 components o Vascular wall (endothelium) o Platelets o Coagulation cascade NORMAL HEMOSTASIS 1. Endothelial injury expose high thrombogenic subendothelial extracellular matrix which facilitates PLATELET ADHERENCE AND ACTIVATION a. Activation of platelets results in dramatic shape change (disc to flat plans with increased surface area) and release secretory granules b. Secreted product recruit additional platelets (AGGREGATION) to form hemostatic plug c. Formation of platelet plug = primary hemostasis 2. Tissue factor exposed at the site of injury (factor III and thromboplastin) a. TF membrane bound procoagulant glycoprotein made by endothelial cells b. Acts with factor 7 as major in vivo initiator of the coagulation cascade, eventually becoming thrombin c. Thrombin cleaves circulating fibrinogen into fibrin  creates fibrin meshwork and induces additional recruitment and activation d. Secondary hemostasis – formation of fibrin plug 3. Polymerized fibrin and platelet aggregate form a solid permanent plug to prevent any hemorrhage a. Counter regulatory mechanisms (like tissue plasminogen activator t-PA) set into motion to limit the hemostasis to site of injury



Endothelium – key players in the regulation of hemostasis  balance between anti and prothrombotic activities determines whether thrombus formation, propagation or dissolution occurs, A. ANTITHROMBOTIC PROPERTIES –normally exhibit antiplatelet, anticoagulant and fibrinolytic properties  actively prevent thrombosis 1. Antiplatelet effects: prevents platelets and plasma coagulation factors from engaging subendothelial ECM a. Nonactivated platelets don’t adhere b. Prostacyclin and NO impede platelet adhesion (vasodilators and inhibitors of platelet aggregation c. Degrade ADP and further inhibit platelet aggregation 2. Anticoagulant effects: mediated by a. Membrane associated heparin like molecules- act indirectly and are cofactors that greatly enhance inactivation of thrombin and anti Thrombin III b. Thrombomodulin – binds to thrombin and converts it into an anticoagulant via ability to activate protein C – inhibits clotting inactivators factors 5a and 7a i. Also protein S (cofactor for protein C) c. TF pathway inhibitor (TFPI) – cells surface protein that direct inhibits TF, factor 7a, and 10a 3. Fibrinolytic effects: make tissue type plasminogen activator (t-PA) protease that cleaves plasminogen to form plasmin, plasmin cleaves fibrin to degrade thrombus B. Prothrombotic properties after injury or activation by infection agents, hemodynamic forces plasma mediator and cytokines  procoagulant activities 1. Platelet effects – injury allows platelets to contact ECM adhesion occurs through interaction with vWF a. Glycoprotein vWF – adhesion bridge between sub endothelial collagen and Gp1 platelet receptor b. Adhesion  fibrinogen connects GpIIb-IIIa receptors on different platelets c. ADP incudes conformational change 2. Procoagulant effects – in response to cytokines (TNF or IL1) or bacterial endotoxin, endothelial cell make TF, the major activator of the extrinsic clotting cascade a. Increase the catalytic function of activated factors 9a and 10a 3. Anti fibrinolytic effects- endothelial cells secrete inhibitors of plasminogen activator (PAIs)_ which limit fibrinolysis to favor thrombosis Platelets A. Description 1. Disc shaped anucleate cell fragments that are shed from megakaryocytes I BM in blood stream 2. From primary hemostatic plug that initially seals vascular defects and provides a surface that recruits and concentrates activated CF 3. Contain a. Glycoprotein receptors b. Contractile skeleton c. 2 types of cytoplasmic granules i. Alpha granules have adhesion molecule P-selectin on membrane  Contain fibrinogen, fibronectin, factors 5 and 8, platelet factor 4, PDGF and TGF beta ii. Dense (delta) granules


 Contain ADP/ATP, calcium, histamine, serotonin and epinephrine B. Encounter ECM constituents like collagen and vWF and contact them to under go 1. Adhesion and shape change a. Platelet adhesion to ECM mediated by interactions by vWF which connects Gp1b (platelet surface receptor) and exposed collagen b. WWF-Gp1b bond necessary to overcome high shear forces of flowing blood c. Genetic deficiencies: vWF (vWF disease) and receptor (Bernard-Soldier syndrome) result in bleeding disorder 2. Secretion (release reaction) of both granules occurs after adhesion a. Initiate intracellular protein phosphorylation cascade leading to degranulation b. Release of dense bodies important since calcium is required in coagulation cascade and ADP is potent activator of platelet aggregation i. ADP also makes ADP release, amplifying process ii. Activation  negatively charged phospholipids (phosphatidyserine) on their surface  Bind calcium and are nucleation site to assemble the complexes containing coagulation factors 3. Aggregation follows adhesion and granule release a. Thromboxane A2 (TxA2) amplifies platelet aggregation which lead to formation of primary hemostatic plug b. Platelet aggregation is reversible, but it activates the coagulation cascade which generates thrombin c. Thrombin stabilizes platelet plug 2 ways i. Binds to protease activated receptor on platelet membrane and causes further platelet aggregation ii. Followed by platelet contraction – dependent on platelet cytoskeleton that creates an irreversible fused mass of platelets which makes up the secondary hemostatic plug iii. Thrombin converts fibrinogen to fibrin – which cements platelets in place 4. Platelet activation by ADP triggers conformational change in platelet GpIIb-IIIa receptors that induces binding to fibrinogen which forms interactions between platelets C. Platelet endothelial cell interactions 1. Prostacyclin (PGI2) from endothelial cells inhibits platelet aggregation and is vasodilator 2. TxA2 is platelet aggregation and vasoconstrictor 3. At baseline, platelet aggregation is prevented 4. Endothelial damage promotes hemostatic plug formation 5. Aspirin – blocks platelet TxA2 synthesis a. PGI2 production inhibited by aspirin, endothelial cells can resynthesize active cyooxyfenase and overcome blockage 6. Like PGi2, NO acts a vasodilator and inhibits platelet aggregation Coagulation cascade A. Description 1. Thrombin ascts at numerous points 2. Each rxn depends on assmelyof ac omplex composed of enzyme, a substrate (proenzyme of next coagulation factor in series) and a cofactor

3. Assembled on pholipid suface and held together by interactions that depend on calcium ions 4. Divided into extrinsic and intriskc and converge at facor X B. Tests 1. Prothrombin time (PT) secreeens for aciivity of the proteins in extrinsisc (7, 10, 2 v and friobrnogen) a. Phsospholipids plus TF to pateint’s citrated plasma, and calcium and measure the time ti takes for fibrin clot to form b. Factor 7 its vitamin k dpeende coagulation factor w/ shortest time 2. Partilal thromboplast time (PTT) screens fro activity in the intrinsic pathways (factors 12, 11, 9, 8, 1, 2, 5,10) a. Phospholipids plus factor 12 to patien’ts plasma, then Ca and measure the time for clot formation (28-35 seconds0 i. Sensitive to heparin Initiation stage: TF located in the vessel wall from ma monocyte or endotheial cell binds factor 7, which ciruculates in plasama  TF-F7a complex cleaves small amounts of both factor 9 and factor 10  Thrombin cleaves factor 8 and 5 to being proprgtaion stage Propgoagtion stage –  Factors 9 and factor 8 convert 10 to 10a  Factor 10a complexds with 5a  Compellx vonvert prothrombin to thrombin  Essential cofactors are cealciumand phospholipids Thrboming is essential  release of factor 8 from vWF and activation aof faa

. B. Platelets bind via glycoprotein Ib (GpIb) receptors to von Willebrand factor (vWF) on exposed extracellular matrix (ECM) and are activated, undergoing a shape change and granule release. Released adenosine diphosphate (ADP) and thromboxane A 2 (TxA 2) induce additional platelet aggregation through platelet GpIIbIIIa receptor binding to fibrinogen, and form the primary hemostatic plug.

C. Local activation of the coagulation cascade (involving tissue factor and platelet phospholipids) results in fibrin polymerization, “cementing” the platelets into a definitive secondary hemostatic plug.

D, Counter-regulatory mechanisms, mediated by tissue plasminogen activator (t-PA, a fibrinolytic product) and thrombomodulin, confine the hemostatic process to the site of injury.


BLEEDING DISORDERS: Can result from 1. Increased fragility of vessels

2. Platelet deficiency or dysfunction 3. Derangement of coagulation
FIGURE 4-8 Coagulation cascade. Factor IX can be activated either by factor XIa or factor VIIa; in lab tests, activation is predominantly dependent on factor XIa of the intrinsic pathway. Factors in red boxes represent inactive molecules; activated factors are indicated with a lower case “a” and a green box. Note also the multiple points where thrombin (factor IIa; light blue boxes) contributes to coagulation through positive feedback loops. The red “X”s denotes points of action of tissue factor pathway inhibitor (TFPI), which inhibits the activation of factors X and IX by factor VIIa. PL, phospholipid; HMWK, high-molecular-weight kininogen.