Blood Clotting

1. Blood Clotting Lecture 8 Dr. Gihan Gawish

2. Coagulation • Coagulation is a complex process by which blood forms clots. • Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium (lining of the vessel).

3. Primary hemostasis: platelets immediately form a plug at the site of injury Secondary hemostasis: occurs simultaneously: proteins in the blood plasma (clotting factors) respond in a complex cascade to form fibrin strands which strengthen the platelet plug Hemostasis

4. 1. Primary hemostasisPlatelet activation • Damage to blood vessel walls exposes sub endothelium proteins, most notably collagen, present under the endothelium. • Circulating platelets bind collagen with surface collagen-specific glycoprotein Ia/IIa receptors.

5. vWF BLOOD PLATELETS ENDOTHELIAL CELL vWF vWF vWF EXPOSED COLLAGEN 3. The adhesion is strengthened by circulating proteins (vWF) Binding intermediaries von Willebrand factor

6. 4. This adhesion activates the platelets. 5. Activated platelets release the contents of stored granules into the blood: • plasma ADP, • serotonin, • platelet activating factor (PAF), • von Willebrand factor (vWF) , • platelet factor 4 • thromboxane A2 (TXA2)

7. 6. The granules' contents activate: Gq-linked protein receptor cascade Increased calcium concentration in the platelets' cytosol. Activates protein kinase C Activates phospholipase A2 (PLA2). Modifies the integrin membrane glycoprotein Increasing its affinity to bind fibrinogen.

8. 7. The activated platelets changed shape from spherical to stellate 8. The fibrinogen cross-links with glycoprotein aid in aggregation of adjacent platelets.

9. 2. Secondary hemostasisThe coagulation cascade • The coagulation cascade of secondary hemostasis has two pathways: • The contact activation pathway (formerly known as the intrinsic pathway) • The tissue factor pathway (formerly known as the extrinsic pathway) That lead to fibrin formation.

10. Thrombin Fibrinpolymers Fibrinmonomers Fibrinogen The Clotting Cascade

11. The biological of the coagulation factors • The coagulation factors are generally serine proteases (enzymes). • There are some exceptions. For example, FVIII and FV are glycoproteins • Factor XIII is a transglutaminase. • Serine proteases act by cleaving other proteins at specific sites. • The coagulation factors circulate as inactive zymogens.

12. The pathways are a series of reactions • zymogen + its glycoprotein co-factor are activated to become active components that then catalyze the next reaction in the cascade, ultimately resulting in cross-linked fibrin. zymogen is inactive enzyme precursor of a serine protease

13. Coagulation factors are generally indicated by Roman numerals, with a lowercase a appended to indicate an active form.

14. Generation of Thrombin • The prothrombin (Factor II) geneis located on the eleventhchromosome(11p11-q12 ( • Thrombin is produced by the enzymatic cleavage of two sites on prothrombin by activated Factor X (Xa). • The activity of factor Xa is greatly enhanced by binding to activated Factor V (Va), termed the prothrombinase complex.

15. Prothrombin is produced in the liver and is post-translationally modified in a vitamin K-dependent reaction that converts ten glutamic acids on prothrombin to gamma-carboxyglutamic acid (Gla). • In the presence of calcium, the Gla residues promote the binding of thrombin to phospholipid bilayers

16. Deficiency of vitamin K or administration of the anticoagulant warfarin inhibits the production of gamma-carboxyglutamic acid residues, slowing the activation of the coagulation cascade. • In human beings the level of prothrombin in the blood stream increases after birth and typically peaks on the 8th day after which the prothrombin level lowers to normal levels

17. Action of Thrombin • Thrombin converts fibrinogen to an active form that assembles into fibrin. • Thrombin also activatesfactor XI, factor V, andfactor VIII. This positive feedback accelerates the production of thrombin. • Factor XIIIis also activated by thrombin. Factor XIIIa is atransglutaminasethat catalyzes the formation of covalent bonds between lysine and glutamine residues in fibrin. The covalent bonds increase the stability of the fibrin clot.

18. Action of Thrombin In platelets • In addition to the thrombin activity in the coagulation cascades, thrombin also promotesplateletactivation, via activation ofprotease-activated receptorson the platelet.

19. Ca

21. Electron Micrograph of Fibrin

22. A fibrin clot is formed by the interplay of the intrinsic, extrinsic, and final common pathways. • The intrinsic pathway begins with the activation of factor XII (Hageman factor) by contact with abnormal surfaces produced by injury.

23. The extrinsic pathway is triggered by trauma, which activates factor VII and releases a lipoprotein, called tissue factor, from blood vessels. Inactive forms of clotting factors are shown in red; their activated counterparts (indicated by the subscript "a") are in yellow. • Stimulatory proteins that are not themselves enzymes are shown in blue. • A striking feature of this process is that the activated form of one clotting factor catalyzes the activation of the next factor. I.

24. Cofactors Various substances are required for the proper functioning of the coagulation cascade: • Calcium and phospholipids (a platelet membrane constituent) • They are required for the tenase and prothrombinase complexes to function.

25. Calcium mediates the binding of the complexes via the terminal gamma-carboxyl residues on • FXa • FIXa (to the phospholipids surfaces expressed by platelets) The Calcium-Binding Region of Prothrombin Prothrombin binds calcium ions with the modified amino acid g-carboxyglutamate (red).

26. 2. Vitamin K • It is an essential factor to a hepaticgamma-glutamyl carboxylasethat adds acarboxyl group toglutamic acid residues on: • Factor II, • Factor VII, • Factor IX • Factor X, • Protein S, • Protein C

27. In adding the gamma-carboxyl group to glutamate residues on the immature clotting factors Vitamin K is itself oxidized.

28. Another enzyme, Vitamin K epoxide reductase VKORCreduces vitamin K back to its active form. • Vitamin K epoxide reductase is pharmacologically important as a target for anticoagulant (antagonists) drugswarfarin and relatedcoumarinssuch asacenocoumarol, phenprocoumonanddicumarol.

29. These drugs create a deficiency of reduced vitamin K by blocking VKORC, thereby inhibiting maturation of clotting factors. • Other deficiencies of vitamin K (e.g. inmalabsorption), or disease (hepatocellular carcinoma impairs the function of the enzyme and leads to the formation of PIVKAs (proteins formed in vitamin K absence) this causes partial or non gamma carboxylation and affects the coagulation factors ability to bind to expressed phospholipids

30. The Clotting Process Must Be Precisely Regulated • There is a fine line between hemorrhage and thrombosis. Clots must form rapidly yet remain confined to the area of injury. • Activated factors are short-lived because they are diluted by blood flow, removed by the liver, and degraded by proteases. • For example, the stimulatory proteins factors Va and VIIIa are digested by protein C

31. Protein C is a protease that is switched on by the action of thrombin. • Thus, thrombin has a dual function: it catalyzes the formation of fibrin and it initiates the deactivation of the clotting cascade.

32. Fibrinolysis

33. Fibrinolysis is the process wherein a fibrin clot is broken down • Plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver. • Plasmin is produced in an inactive form, plasminogen, in the liver.

34. Coagulation factors and related substances • I fibrinogen Forms clot (fibrin( • (II (prothrombin Its active form (IIa) activates I, V, VII, VIII, XI, XIII, protein C, platelets • Tissue factor Co-factor of VIIa (formerly known as factor III) • Calcium Required for coagulation factors to bind to phospholipid (formerly known as factor IV • )V (proaccelerin, labile factor )Co-factor of X with which it forms theprothrombinase complex

35. XII (Hageman factor) Activates factor XI and prekallikrein • XIII (fibrin-stabilizing factor) Cross links fibrin • von Willebrand factor Binds to VIII, mediates platelet adhesion • Prekallikrein Activates XII and prekallikrein; cleaves HMWK • high molecular weight kininogen (HMWK) Supports reciprocal activation of XII, XI, and prekallikrein

36. antithrombinIII Inhibits IIa, Xa, and other proteases • heparin cofactor II Inhibits IIa, cofactor for heparin • protein C Inactivates Va and VIIIa • protein S Cofactor for activated protein C

37. Specific inhibitors of clotting factors 1.Antithrombin III is the most important one, • It is a plasma protein that inactivates thrombin by forming an irreversible complex with it. • It resembles alpha 1-antitrypsin except that it inhibits thrombin much more strongly than it inhibits elastase. • Also, it blocks other serine proteases in the clotting cascade namely, factors XIIa, XIa, IXa, and Xa.

38. 2.Heparin • The inhibitory action of antithrombin III is enhanced by heparin • It is a negatively charged polysaccharide found in mast cells near the walls of blood vessels and on the surfaces of endothelial cells • Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors. • Antitrypsin and antithrombin are serpins, a family of serine protease inhibitors.

39. Electron Micrograph of a Mast Cell. Heparin and other molecules in the dense granules are released into the extracellular space when the cell is triggered to secrete.

40. 3. Alpha 1-antitrypsin • which normally inhibits elastase • alpha 1-Antitrypsin activity normally increases markedly after injury to counteract excess elastase arising from stimulated neutrophils. • The mutant a 1-antitrypsin caused the patient's thrombin activity to drop to such a low level that hemorrhage ensued.

41. Disease and clinical significance of thrombosis 1. Hemophilias are the best-known coagulation factor disorders. The three main forms are: hemophilia C (factor XI deficiency, mild bleeding tendency). hemophilia A (factor VIII deficiency) hemophilia B (factor IX deficiency or "Christmas disease")

42. 2. von Willebrand disease • It is the most common hereditary bleeding disorder and is characterized as being inherited autosomal recessive or dominant. • In this disease there is a defect in von Willebrand factor (vWF) which mediates the binding of glycoprotein Ib (GPIb) to collagen. • This binding helps mediate the activation of platelets and formation of primary hemostasis.

43. 3. Deficiency of Vitamin K • It may also contribute to bleeding disorders because clotting factor maturation depends on Vitamin K. 4. Liver diseases: • Some clotting factors; II, IX, VII, X are synthesized in liver • Liver diseases deficiency of these factors bleeding disorders.

44. Coagulation Tests

45. Coagulation Cascade PT VIIIa PTT Heparin Hirudin, Argatroban

46. Coagulation and Fibrinolysis

47. Screen test Bleeding Time (Duke method, Template method), Thrombin Time, PT, PTT Antiphospholipid syndrome Dilute Russell Viper Venom Time (dRVVT), Anti-Cardiolipin Ab, ACA, IgG, Anti-Phospholipid Ab, APA, IgG, Anti-Cardiolipin Ab, ACA, IgM Coagulation factor Factor I (Fibrinogen), II, V, VII, VWF, VIII, IX, X, Urea solubility test, Other coagulation inhibitor Study, VIII, XI, XII DIC profile Fibrinogen, FDP, 3P Test, D-dimer Fibrinolysis Euglobulin clot lysis time, Plasminogen activator inhibitor, Alpha2-antiplasmin PLT function Platelet aggregation Thrombosis APC Resistance, Protein S, Antithrombin III, Protein C, Plasminogen Coagulation Tests

48. 1. Bleeding time • Done with a template. • Time taken for the blood to stop • Normal range 2-10mts • Prolonged in plt disorders, low plts, severe anemia, Vwf, collagen vascular disease • Great variability in results, unreliable, invasive, insensitive

49. 2. Activated Partial Thromboplastin Time (aPTT) • It is a performance indicator measuring the efficancy of both the "intrinsic" and the common coagulation pathways. • It is also used to monitor the treatment effects with heparin. • It is used in conjunction with the prothrombin time (PT) which measures the extrinsic pathway.

50. Methodology (aPTT) • Container: blue top (3.2% citrate) tube • Collection: Deliver tubes immediately to the laboratory • In order to activate the intrinsic pathway, phospholipid, an activator (such as silica, celite, kaolin, ellagic acid), and calcium (to reverse the anticoagulant effect of the citrate) are mixed into the plasma sample . • The time is measured until a thrombus (clot) forms. • The test is termed "partial" due to the absence of tissue factor from the reaction mixture.