1. Fibrinolysis, anticaogulants, related aspects

2. Learning objectives Learning objectives Clot retraction Clot retraction Fibrinolysis Fibrinolysis Thromboses, emboli. Thromboses, emboli. Anticoagulants- in-vitro, in-vivo and their mechanism of actions Anticoagulants- in-vitro, in-vivo and their mechanism of actions Consequences of deficiencies of platelets & clotting factors Consequences of deficiencies of platelets & clotting factors Tests- clotting time, bleeding time, prothrombin time, activated patial thromboplastin time. Tests- clotting time, bleeding time, prothrombin time, activated patial thromboplastin time.

3. CLOT RETRACTION Def - consolidation or tightening of clot Def - consolidation or tightening of clot Method - fibrin threads contract/shrink Method - fibrin threads contract/shrink Benefits - pull damaged edges of vessel wall together, repair is faster; tighter, stronger clot Benefits - pull damaged edges of vessel wall together, repair is faster; tighter, stronger clot Timing - retraction occurs within 30-60 min. Timing - retraction occurs within 30-60 min.

6. FIBRINOLYSIS Dissolution of Clot Dissolution of Clot Control Control thrombin gets trapped inside the clot thrombin gets trapped inside the clot thrombin converts inactive plasminogen in the clot into active plasmin thrombin converts inactive plasminogen in the clot into active plasmin Plasmin Plasmin active form of the enzyme digests fibrin threads active form of the enzyme digests fibrin threads important in removal of small clots in small blood vessels important in removal of small clots in small blood vessels Timing Timing very slowly compared to clot formation very slowly compared to clot formation i.e after tissue repair of vessel wall

7. Thrombomodulin+ thrombinendothelium Protein CActivated protein C Inactivation of inhibitors Of t-PA PlasminogenPlasmin t-PA Fibrin FDP Thrombin, u-PA, t-PA FIBRINO LYSIS

8. USE OF FIBRINOLYTIC DRUGS example - "tissue plasminogen factor" example - "tissue plasminogen factor" drug is given in active form for fast breakdown of abnormal clots drug is given in active form for fast breakdown of abnormal clots clots that cause heart attacks & some brain attacks or strokes clots that cause heart attacks & some brain attacks or strokes drugs quickly break up clots & restore blood flow drugs quickly break up clots & restore blood flow increases survival rates among these patients & reduces disability increases survival rates among these patients & reduces disability

9. What is Thrombosis ? What is Thrombosis ? Formation of clots inside the blood vessels is called as thrombosis to distinguish it from normal extravascular clotting of blood. Formation of clots inside the blood vessels is called as thrombosis to distinguish it from normal extravascular clotting of blood. Bits of thrombus some times break off from the site and travel in blood to different site called as EMBOLI Bits of thrombus some times break off from the site and travel in blood to different site called as EMBOLI

11. ROLE OF VIT ‘K’ Synthesis of clotting factors (II - prothrombin, VII, IX, X, Protein C, Protein S) VII, IX, X, Protein C, Protein S) lipid soluble - bile is necessary for it’s absorption factors that impair Vit. K availabilty are liver damage or blocked bile ducts liver damage or blocked bile ducts prolonged use of antibiotics (kills off GI bacterial flora) prolonged use of antibiotics (kills off GI bacterial flora) poor intestinal absorption poor intestinal absorption

12. ANTIHEMOSTATIC DRUGS TYPES: 1. BY PRVENTING PLATELET AGGREGATION 1. BY PRVENTING PLATELET AGGREGATION 2. ANTICOAGULANTS- BY PREVENTING COAGULATION 2. ANTICOAGULANTS- BY PREVENTING COAGULATION 3. THROMBOLYTICS- BY LYSING FIBRIN 3. THROMBOLYTICS- BY LYSING FIBRIN

13. Eg; ASPIRIN Eg; ASPIRIN INHIBITS PROSTAGLANDINS & THROMBOXANE A2, SO PREVENTS PLATELET AGGREGATION INHIBITS PROSTAGLANDINS & THROMBOXANE A2, SO PREVENTS PLATELET AGGREGATION

14. FACTORS OPPOSING CLOTTING 1. TFPI BY ENDOTHELIUM INHIBITS EXTRINSIC PATHWAY 1. TFPI BY ENDOTHELIUM INHIBITS EXTRINSIC PATHWAY 2. THROMBIN+ THROMBOMODULIN ON ENDOTHELIUM BY ACTIVATING PROTEIN C+S INHIBIT FACTORS VIIIa & Va 2. THROMBIN+ THROMBOMODULIN ON ENDOTHELIUM BY ACTIVATING PROTEIN C+S INHIBIT FACTORS VIIIa & Va

15. 3. ANTITHROMBIN III INACTIVATES THROMBIN & OTHER CLOTTING FACTORS (IX, X, XI, XII) VIA BINDING TO HEPARIN 3. ANTITHROMBIN III INACTIVATES THROMBIN & OTHER CLOTTING FACTORS (IX, X, XI, XII) VIA BINDING TO HEPARIN 4. DEFECIENCY OF VIT K BY PREVENTING GAMMA CARBOXYLATION OF FACTORS II, VII, IX, X, PROTEIN C, PROTEIN S 4. DEFECIENCY OF VIT K BY PREVENTING GAMMA CARBOXYLATION OF FACTORS II, VII, IX, X, PROTEIN C, PROTEIN S

16. ANTICOAGULANTS Prevents clotting Prevents clotting Risk: High doses may cause excessive bleeding Risk: High doses may cause excessive bleeding 1. IN-VIVO egs:heparin, dicumarol, warfarin 1. IN-VIVO egs:heparin, dicumarol, warfarin clinically used in individuals at risk of pathologic clotting such as those with coronary artery disease (with endothelial injury) clinically used in individuals at risk of pathologic clotting such as those with coronary artery disease (with endothelial injury) 2. IN-VITRO egs: oxalates, citrates, EDTA, heparin. 2. IN-VITRO egs: oxalates, citrates, EDTA, heparin.

17. In-vivo:heparin stops conversion of prothrombin to thrombin by binding to antithrombin III & activating it, inhibits factors- IXa, Xa, XIa, XIIa stops conversion of prothrombin to thrombin by binding to antithrombin III & activating it, inhibits factors- IXa, Xa, XIa, XIIa abundant in liver and lungs where blood flow is normally slow abundant in liver and lungs where blood flow is normally slow secreted by basophils secreted by basophils Dicumarol - antagonist to Vitamin K, inhibits gamma carboxylation of II, VII, IX, X. Dicumarol - antagonist to Vitamin K, inhibits gamma carboxylation of II, VII, IX, X.

18. IN-VITRO: IN-VITRO: OXALATES, CITRATES, EDTA: OXALATES, CITRATES, EDTA: BY FORMING SALTS OF CALCIUM, BINDING WITH CALCIUM BY FORMING SALTS OF CALCIUM, BINDING WITH CALCIUM

19. THROMBOLYTICS BY CAUSING FIBRINOLYSIS (dissolves clot, re-establishes blood flow in case of blocks). BY CAUSING FIBRINOLYSIS (dissolves clot, re-establishes blood flow in case of blocks). Eg: recombinant t-PA, streptokinase, t- urokinase Eg: recombinant t-PA, streptokinase, t- urokinase

20. Defects in Fibrinolytic System Excessive fibrinolysis Plasminogen activator inhibitor (PAI) deficiency Plasminogen activator inhibitor (PAI) deficiency Impaired clearance of TPA as in chronic liver disease Impaired clearance of TPA as in chronic liver disease Diagnosis: low fibrinogen levels, ↑ fibrinogen degradation products in plasma Diagnosis: low fibrinogen levels, ↑ fibrinogen degradation products in plasma Consequence: increased likelihood of bleeding Consequence: increased likelihood of bleeding 20

21. Reduced fibrinolysis Excess of plasminogen activator inhibitor Excess of plasminogen activator inhibitor Defects in fibrinogen Defects in fibrinogen Consequence: increased risk of stroke Consequence: increased risk of stroke

22. REASONS FOR ABNORMAL CLOTS Rough Endothelium Rough Endothelium cholesterol/fatty deposits on walls of vessels attract platelets cholesterol/fatty deposits on walls of vessels attract platelets Slower than Normal Blood Flow Slower than Normal Blood Flow clotting factors build up clotting factors build up inactivity inactivity Having prolonged bed rest (several days at a time) Having prolonged bed rest (several days at a time) poor leg muscles, that don't move blood in veins poor leg muscles, that don't move blood in veins narrowing of vessels narrowing of vessels high blood viscosity - slowing blood flow high blood viscosity - slowing blood flow

23. CLOTTING DISORDERS Hemophilia Hemophilia Types – Types – Hemophilia A or classic- VIII Hemophilia A or classic- VIII Hemophilia B- IX Hemophilia B- IX Von will brand disease Inadequate Fibrinogen Inadequate Fibrinogen genetic defect in some cases genetic defect in some cases Due to vit K deficiency Due to vit K deficiency Gross def of any factors mostly congenital Gross def of any factors mostly congenital

24. Laboratory tests for screening hemostasis Platelet count (< 150000/mm 3 called thrombocytopenia) Platelet count (< 150000/mm 3 called thrombocytopenia) Bleeding time Bleeding time Clotting time Clotting time Further tests based on results of above tests. Further tests based on results of above tests. If clotting time is prolonged, then 2 more tests are done: 1. Prothrombin time (PT), screens the extrinsic and the common pathway. 2. Activated partial thromboplastin time (aPTT), this screens the intrinsic + common pathway.

25. Tests for hemostasis contd Clot retraction- 30-60 min Clot retraction- 30-60 min Bleeding time: Bleeding time: Time taken for bleeding to cease from the time of infliction of a standard wound Time taken for bleeding to cease from the time of infliction of a standard wound Normally: 2-6 min, varies with method Normally: 2-6 min, varies with method ‘Bleeding Time’ reflects platelet function, and integrity of vessel wall ‘Bleeding Time’ reflects platelet function, and integrity of vessel wall Causes of prolonged “bleeding time”: Causes of prolonged “bleeding time”: Thrombocytopenia Thrombocytopenia Von Willebrand’s disease Von Willebrand’s disease

26. Clotting time Time from blood taken into a standard capillary tube to form fibrin threads (clot) Time from blood taken into a standard capillary tube to form fibrin threads (clot) Note – clotting time is measured in vitro Note – clotting time is measured in vitro Normally, it ranges from 3-8 min Normally, it ranges from 3-8 min Causes of prolongation of Clotting Time: Causes of prolongation of Clotting Time: Vitamin K deficiency due to any cause Vitamin K deficiency due to any cause Deficiency of specific clotting factors such as hemophilia A (clotting factor VIII deficiency) Deficiency of specific clotting factors such as hemophilia A (clotting factor VIII deficiency) Gross deficiency of other clotting factors Gross deficiency of other clotting factors

27. If clotting time is prolonged, then further testing TestAssessing Factors assessed Prothrombin time- normal - 12-16sec Extrinsic pathway + Common pathway Tissue Factor, VII, X, Prothrombin, Fibrinogen aPTT Intrinsic pathway + Common pathway XII, XI, IX, VIII, X, V, Prothrombin, Fibrinogen 27

28. If PT is normal and aPTT is prolonged, then what? Something wrong with the ‘Intrinsic Pathway’ Something wrong with the ‘Intrinsic Pathway’ Then do specific assay for Factor VIII Then do specific assay for Factor VIII If Factor VIII levels are normal, then Factor IX If Factor VIII levels are normal, then Factor IX Factor VIII deficiency, or factor IX deficiency are common inherited causes of prolongation of aPTT Factor VIII deficiency, or factor IX deficiency are common inherited causes of prolongation of aPTT 28

29. Some facts about vWF and factor VIII: Some facts about vWF and factor VIII: In plasma, factor VIII is normally bound to vWF; In plasma, factor VIII is normally bound to vWF; When there is a deficiency of vWF, factor VIII is lost easily in the urine and deficiency of factor VIII can result When there is a deficiency of vWF, factor VIII is lost easily in the urine and deficiency of factor VIII can result The result – ‘bleeding time’ is prolonged as a result of vWF deficiency, and ‘clotting time’ is prolonged as a result of Factor VIII deficiency. The result – ‘bleeding time’ is prolonged as a result of vWF deficiency, and ‘clotting time’ is prolonged as a result of Factor VIII deficiency.

30. Blood Clotting – Physiologic or Pathologic? What is thrombosis and how does it differ from hemostasis? What is thrombosis and how does it differ from hemostasis? 30 Physiologic Clotting [Hemostasis] Pathologic Clotting [Intravascular Clotting or Thrombosis] Stimulus – sudden injury of blood vessel resulting in bleeding into tissues Stimulus – chronic endothelial injury (atherosclerotic plaques) in otherwise intact blood vessels; i.e., typically, there is no bleeding from the vessel. Response – Clotting at the site of injury minimizes blood loss Response – clotting at the site narrows the lumen of the blood vessel inhibiting flow downstream; example – it could result in a heart attack.

31. Consequences of an Excess of Clotting Factors or “Inappropriate Activation” of the Clotting Cascade – It depends on where it happens.. Thrombosis in coronary artery /arteries Decrease in blood flow to cardiac muscle cells supplied by the narrowed coronary artery If sustained Death of cardiac muscle cells supplied by this artery (myocardial infarction, commonly called heart attack) 31

32. Mechanisms that put an individual at risk of inappropriate clotting (thrombosis) Sluggish blood flow at a particular site (example, deep veins of the legs during prolonged immobilization due to any reason can lead to thrombosis in deep veins, DVT) Sluggish blood flow at a particular site (example, deep veins of the legs during prolonged immobilization due to any reason can lead to thrombosis in deep veins, DVT) Endothelial injury due to any cause (chronic cigarette smoking, hypertension etc, diabetes ) Endothelial injury due to any cause (chronic cigarette smoking, hypertension etc, diabetes ) 32

33. Inherited Single Gene Mutations that result in hypercoagulable states (less common) Deficiency of protein C Deficiency of protein C Deficiency of protein S Deficiency of protein S Deficiency of antithrombin III Deficiency of antithrombin III Presence of factor V Leiden (relatively more common than the other three above) Presence of factor V Leiden (relatively more common than the other three above)

34. What is Factor V Leiden? It is Factor V that is resistant to inhibition by activated protein C. It is Factor V that is resistant to inhibition by activated protein C. Consequence: unrestrained clotting with the risk of inappropriate clotting (thrombosis) Consequence: unrestrained clotting with the risk of inappropriate clotting (thrombosis) 34

35. Learning outcomes Learning outcomes At the end of this class student should be able to answer the following At the end of this class student should be able to answer the following How the clot is lysed ? What is the functional significance of this ? How the clot is lysed ? What is the functional significance of this ? What is meant by clot retraction? What is the underlying mechanism ? What is meant by clot retraction? What is the underlying mechanism ? What is thromboses ? What is emboli ? What is thromboses ? What is emboli ? List the in vivo and in vitro anticoagulants List the in vivo and in vitro anticoagulants Describe the mechanism of action of the following anticoagulants; heparin, warfarin, potassium oxalate, ammonium oxalate. Describe the mechanism of action of the following anticoagulants; heparin, warfarin, potassium oxalate, ammonium oxalate.

36. Regarding bleeding time; normal, mechanism of stoppage, causes of prolonged BT Regarding bleeding time; normal, mechanism of stoppage, causes of prolonged BT Regarding clotting time; normal, mechanisms involved,when is it prolonged Regarding clotting time; normal, mechanisms involved,when is it prolonged Clotting disorders and causes Clotting disorders and causes What is prothrombin time ? What activated partial thromboplastin time ? Purpose of these tests ? What is prothrombin time ? What activated partial thromboplastin time ? Purpose of these tests ?