HEMOSTAZA

1. HEMOSTAZA Curs an IV limbaengleza 2012-2013

2. HEMOSTASIS Hemostasisis defined as a property of circulation whereby blood is maintained within a vessel and the ability of the system to prevent excessive blood loss when injured. Process is rapid and localized

3. HEMOSTASIS • Hemostasis can be divided into two stages • Primary hemostasis • Response to vascular injury • Formation of the “platelet plug” adhering to the endothelial wall • Limits bleeding immediately • Secondary Hemostasis • Results in formation of a stable clot • Involves the enzymatic activation of coagulation proteins that function to produce fibrin as a reinforcement of the platelet plug • Gradually the stable plug will be dissolved by fibrinolysis

4. HEMOSTASIS • Following an injury to blood vessels several actions may help prevent blood loss, including: Formation of a clot

5. HEMOSTASIS • The primary players in hemostasis include • Blood vessels • Platlets • Plasma proteins • Coagulation proteins – involved in clot formation • Fibrinolysis – involved in clot dissolution • Serine protease inhibitors • Other minor players include • Kinin system • Complement system

6. VASCULAR SYSTEM • Smooth and continuous endothelial lining is designed to facilitate blood flow • Intact endothelial cells inhibit platelet adherence and blood coagulation • Injury to endothelial cells promotes localized clot formation • Vasoconstriction • Narrows the lumen of the vessel to minimize the loss of blood • Brings the hemostatic components of the blood (platelets and plasma proteins) into closer proximity to the vessel wall • Enhances contact activation of platlets • Von Willebrand factor • Collagen fibers • Platlet membrane glycoprotein Ib • Activated platlets enhance activation of coagulation proteins

7. LOCALVASOCONSTRICTION • is due to local spasm of the smooth muscle (symp. reflex) • can be maintained by platelet vasoconstrictors

8. PRIMARY HEMOSTASIS • Platelets • Interact with injured vessel wall • Interact with each other • Produce the primary hemostatic plug • Primary platelet plug • Fragile • Can easily be dislodged from the vessel wall

9. PLATLET FUNCTION • Provide negatively charged surface for factor X and prothrombinactivation • Release substances that mediate vasoconstriction, platlet aggregation, coagulation, and vascular repair • Provide surface membrane proteins to attach to other platlets, bind collagen, and subendothelium

10. ADHESION Damage to endothelium exposes blood to the subepithelial tissue matrix with adhesive molecules Platlet receptor GPIb binds to subendothelium collagen fibers through von Willebrand’s factor (vWF) Platlet adherence stops the initial bleeding

11. ADHESION

12. SHAPE CHANGE Following vessel injury and platlet exposure to external stimuli, platlets change shape from circulating discs to spheres with pseudopods Exposure of platlet membrane phospholipids promotes the assembly of vitamin-K dependent factors on the platlet membrane surface Activated platlets adhere to exposed collagen

13. AGGREGATION • Platlet-to-platlet interaction • Begins 10-20 seconds after vascular injury and platlet adhesion • Requires dense granule release from the adhering platlets • Requires Ca++and ATP • Requires fibrinogen and fibrinogen receptors GPIIb and IIIa • Mechanism: • ADP released from platlet cytoplasm upon adherence induces exposure of fibrinogen receptors GPIIb and IIIa • Fibrinogen binds to the exposed GPIIb and IIIa • Extracellular Ca++-dependent fibrinogen bridges form between adjacent platlets, thereby promoting platlet aggregation • This is primary or reversible aggregation • Secondary aggregation begins with the release of dense granules • Secondary aggregation is considered irreversible

14. SECRETION Secondary aggregation begins with platlet secretion of dense granules Dense granules contain large amounts of ADP ADP binds to the platlet membrane triggering the synthesis and release of TXA2 The release of large amounts of ADP combined with TXA2 amplifies the initial aggregation of platlets into a large platlet mass

15. PRIMARY HEMOSTASIS

16. SECONDARY HEMOSTASIS • Also called the fibrin forming coagulation system • The purpose is toreinforce the platlet plug • System mediated by many coagulation factors present in an inactive form in blood. • Factors are assigned Roman numerals, I through XIII • Assigned in order of discovery, not place in reaction sequence • Each one has one or more common names or synonyms • Roman numeral followed by letter “a” indicates the activated form of the factor • All are produced in the liver. The von Willebrand factor is also produced in endothelial cells and megakaryocytes.

17. SECONDARY HEMOSTASIS • The cascade theory of blood coagulation • Involves a series of biochemical reactions • Transforms circulating substances into an insoluble gel through conversion of fibrinogen to fibrin • Requires • Plasma proteins • Phospholipids • calcium

18. COAGULATION - CASCADE THEORY Each coagulation factor is converted to an active form by the preceeding factor in the cascade Calcium participates in some of the reactions as a co-factor The blood coagulation cascade occurs on cell surface membranes.

19. COAGULATION - CASCADE THEORY • Clotting factors bind to the phospholipid membrane surface and rearrange until a complex including enzyme, substrate, and cofactor is formed. • Subendothelial tissue exposed with vascular injury and platlet surface provide the critical membranes • The membrane localizes the reaction to the site of injury

20. SECONDARY HEMOSTASIS • Three different complexes assemble on the phospholipid membrane • The pathways for the formation of these complexes are • Intrinsic • Extrinsic • Common -Both intrinsic and extrinsic pathways converge to share factors in the common pathway • Both intrinsic and extrinsic pathways require initiation • Intrinsic - all factors involved in clot formation are in the vascular compartment • Extrinsic- is initiated when a tissue factor not found in blood enters the vascular system

21. EXTRINSIC PATHWAY The extrinsic pathway is initiated when there is an injury to a blood vessel wall In the presence of the tissue factor released from the injured nonvascular tissue (factor III or thromboplastin) and calcium (factor IV), factor VII is activated to factor VIIa Factor VIIa activates factor X to Xa Factor VIIa can also activate factor IX in the intrinsic pathway to IXa

22. EXTRINSIC PATHWAY

23. INTRINSIC PATHWAY Is initiated following exposure to negatively charged foreign substances such as collagen, subendothelium, or phospholipids. Factor XII is activated to XIIa. XIIa then interacts with the contact factors, prekallikrein and kininogen, to activate Factor XI to XIa XIa then activates Factor IX to IXa in the presence of Ca++ IXa participates along with co-factor VIII:C, Ca++, and PF3 (a source of phospholipids), to activate Factor X which leads to the generation of thrombin IXa -factor VIIIa-phospholipid-Ca++ is called the tenase complex

24. INTRINSIC PATHWAY The complex of IXa, VIIIa, X, PF3,and Ca++ assembles on the surface of activated platlets (supply the phospholipid). The surface provides the protective environment for the enzymatic reactions to occur In plasma, VIII circulates as a complex in association with C which has the procoagulant activity and von Willebrand factor (vWF) which functions as a carrier protein. VIII requires enhancement by the generated enzyme thrombin to amplify its activity

25. Intrinsic Pathway PTT Partial Thromboplastin Time XIIXIIa XI XIa IXIXa VIIIa+Ca+Pl X Xa Va+Ca+Pl IIIIa Fibrinogen Fibrin

26. COMMON PATHWAY • Intrinsic and extrinsic pathways • Converge on the common pathway • Both pathways activate Factor-X to Xa • Xa in the presence of Factor V, Ca++ and phospholipid converts prothrombin (Factor II) to its active form thrombin (IIa) • Thrombin then feeds back to activate factors VIII and V, converts fibrinogen to soluble fibrin, and helps to stabilize the fibrin monomer by converting factor XIII to XIIIa. • XIIIa cross-links the fibrin monomers to form a stable fibrin polymer

27. FIBRINOGEN • 3 stages of conversion of fibrinogen to fibrin • Proteolysis • Thrombin cleavage of fibrinogen results in fibrin monomers • Polymerization • Spontaneous self-assembly into fibrin polymers • Stabilization • Introduction of covalent bonds into fibrin polymers by XIIIa

28. COAGULATION

29. THE CLOT

30. FIBRINOLYSIS • The last stage of coagulation isfibrinolysis, which is the dissolution and localization of a fibrin clot. • Prevents excessive fibrin deposition • Allows closely coupled with fibrin formation • Localized surface bound phenomenon that is catalyzed by fibrin formation

31. FIBRINOLYSIS • Fibrinolytic system is a complement to the coagulation system – a fine balance between the two systems must occur • Restricts fibrin formation to area of injury • Dissolves clot by digestion of fibrin • Initiated when coagulation begins – will ultimately dissolve clot • plasminogen ↓ • Fibrin plasmin → fibrin degradation products (FDPs)

32. FIBRINOLYSIS - COMPONENTS • Plasminogen -> plasmin • Plasminogen activators • Inactivators of plasminogen • Inhibitors of plasmin

33. FIBRINOLYSIS • Plasminogen is activated and converts to plasmin by factor XII, HMWK,and PK • Plasmin= enzyme which dissolves fibrin clots into protein fragments that are cleared from plasma by the liver • Fibrin degradation products are breakdown fragments of fibrin or fibrinogen. • The protein fragments are designated X, Y, D, and E • Fragments are strong inhibitors of further coagulation by • interfering with the action of thrombin • interfering with platelet aggregation

34. FIBRINOLYSIS • Inhibitors of fibrinolysis = antiplasmins. • Used to regulate and limit plasmin activity and fibrinolysis • alpha-2-antiplasmin • alpha-2-macroglobulin

36. FIBRINOLYSIS

37. FIBRINOLYSIS

38. HEMOSTATIC BALANCE • The regulation of hemostatic and fibrinolytic processes is dynamic • Balance between • Pro- and anti-hemostatic mediators • Pro- and anti-fibrinolytic mediators • Balance can be upset if any components are • Inadequate • Excessive • Development of thrombi • Excessive local or systemic activation of coagulation • Sustained bleeding • Excessive local or systemic fibrinolytic activity

39. ANTICOAGULANTS • Although tissue breakdown and platelets destruction are normal events in the absence of trauma, intravascular clotting does not usually occur because: • the amounts of procoagulants released are very small • natural anticoagulants are present (Antithrombin III, Heparin, Antithromboplastin, Protein C and S, fibrin fibers)

40. NATURAL ANTICOAGULANTS • Antithrombin III – inhibits factor X and thrombin • Heparinfrom basophils and mast cells potentiates effects of antithrombin III (together they inhibit IX, X, XI, XII and thrombin) • Antithromboplastin (inhibits „tissue factors” – tissue thromboplastins) • Protein C and S – activated by thrombin; degrade factor Va and VIIIa

41. HEMOSTATIC BALANCE • When hemostasis is delayed • Either platelet disorder or a coagulation defect • Bleeding episode may be prolonged • Imbalance created between • An abnormally slow hemostatic rate • A normal rate of fibrinolysis • An inadequate fibrinolytic response • May retard lysis of a thrombus and even contribute to its extension

42. LABORATORY EVALUATION OF HEMOSTASIS Bleeding disorders present differently depending upon the causative problem Platlet disorders present as petechiae and bleeding into mucous membranes because of failure to form the platlet plug Patients with coagulation defects may develop deep spreading hematomas and bleeding into the joints with evident hematuria because of failure to reinforce the platlet plug.

43. LABORATORY EVALUATION • Three different categories of disorders may be found • Vascular and platlet disorders • Coagulatioin factor deficiencies or specific inhibitors • Fibrinolyticdisorders

44. LABORATORY EVALUATION • Tests to differentiate between these include • Platlet count • Peripheral blood smear evaluation • Ivy bleeding time (N=2.5-9.5 min) or platlet function analyzer (PFA) • Prothrombin time (PT) – test contains thromboplastin and calcium chloride and measures measures the extrinsic and common pathways (Normal=11-13 sec)

45. LABORATORY EVALUATION • Activated partial thromboplastin time (APTT) -contact activators and a platlet substitute and calcium chloride are added to measure the intrinsic and common pathways (Normal usually 23-35 sec, may vary depending upon analyzer used, reagents used, and patient population) • Thrombin time (TT) – add thrombin and measure the time required for thrombin to convert fibrinogen to fibrin (common pathway) (N=15-22 sec)

46. LABORATORY EVALUATION • Mixing studies with PT and APTT abnormal results -patient plasma is mixed with normal plasma to distinguish between factor deficiencies and coagulation inhibitors • If assay is corrected – due to factor deficiency • If partially corrected or uncorrected – due to inhibitor • Coagulation factor assays • Assays for fibrin degradation products – evidence of fibrinolysis