Hemostasis

Hemostasis, Hemostats

1. Dr.Niti Sarawgi II MDS HAEMOSTASIS

2. Contents Introduction Events in haemostasis Intrinsic pathway & extrinsic pathway Mechanical haemostasis Chemical haemostasis Thermal haemostasis Conclusion

3. INTRODUCTION Hemostasis is the process of forming clots in the walls of damaged blood vessels and preventing blood loss while maintaining blood in the fluid state within the vascular system.

4. VIRCHOWS TRIAD STASIS OF BLOOD HYPERCOAGULABILITY ENDOTHELIAL INJURY

5. EVENTS IN HAEMOSTASIS Haemostasis means prevention of ‘ Blood Loss ’ . Haemostasis is achieved by several mechanism:- Vascular constriction Formation of platelet plug Formation of blood clot Growth of fibrous tissue into the clot.

6. VASCULAR CONSTRICTION The contraction results from:- Local myogenic spasms Local autacoid factors Nervous reflexes Platelets release, thromboxane-A 2 which is responsible for vasoconstriction of smaller vessels. The more severely a vessel is traumatized, the greater the degree of vascular spasm.

7. Mechanism Vasoconstriction is as a result of increased Ca ion conc. in smooth muscles. Hormonal components : Circulating epinephrine & activation of sympathetic nervous system. Interact with cell surface adrenergic receptors Signal transduction Increased intercellular Ca from sarcoplasmic reticulum Ca-calmodulin complex Activates myosin light- chain kinase stimulate cross bridge cycle

8. Factors : -Exogenous – temperature (cold) -Endogenous-Autonomic nervous system, hormones and mechanisms inherent to vasculature (myogenic response) -Medications: antihistamines , amphetamines , cocaine

9. FORMATION OF PLATELET PLUG PHYSICAL AND CHEMICAL CHARACTERISTICS OF PLATELETS MECHANISM OF PLATELET PLUG Platelet adhesion Platelet activation Platelet aggregation Formation of temporary haemostatic plug

10. NORMAL ARCHITECTURE OF A BLOOD VESSEL

11. PLATELETS Platelets are enucleate cells 1-4 micro meters in size Normal blood concentration of platelets is 1.5L-3L/micro liters Formed in bone marrow from megakaryocytes T-1/2 is 8-12 days Eliminated from the circulation by tissue macrophages.

12. CONTD… Platelet cytoplasm contains active factors such as:- Actin and myosin molecules Thrombosthenin is another contractile protein which can cause platelets to contract. ER and the Golgi apparatus Mitochindria and enzyme system.

13. PLATELET-CELL MEMBRANE Surface coat of glycoproteins a) repulse adherence to normal endothelium b) but adherence to injured vessel wall i.e., endothelial cells, and even more to the exposed collagen from deep with in the vessel wall Large amounts of phospholipids – activates multiple stages in blood-clotting process.

14. PLATELET ADHESON

15. PLATELET ACTIVATION

16. PLATELET AGGREGATION The activated sticky platelets stick to each other form Platelet aggregation TXA2 powerful vasoconsrictor &mobilisation Ca from dense tubular system –activates myosin -actin cause release reaction –Platelet aggregation This involves a series of self sustaining events Leads to formation of platelet plug

17. MECHANISM OF PLATELET PLUG Platelet repair of vascular openings is based on several important functions of the platelet itself Contractile proteins adhere to collagen & vWF Secrete ADP and their enzymes form thromboxane-A2 ADP + thromboxane-A2 activates adjacent platelets Initial Platelet Plug

18. BLOOD COAGULATION IN RUPTURED VESSEL Third mechanism for hemostasis is formation of blood clot Clot begins to develop- severe trauma-15 to 20 sec minor trauma-1 to 2 min

19. CLOTTING PROCESS IN A TRAUMATIZED BLOOD VESSEL

20. MECHANISM OF BLOOD COAGULATION

21. GENERAL MECHANISM In response to rupture of the vessel or damage to the blood itself-formation of prothrombin activator Prothrombin activator catalyzes conversion of prothrombin to thrombin Thrombin catalyzes fibrinogen into fibrin fibers.

22. CONVERSION OF PROTHROMBIN TO THROMBIN

23. PROTHROMBIN & THROMBIN Prothrombin is a plasma protein, an alpha 2 -globulin MW-68700 Plasma concentration- 15mg/dl Unstable protein, splits easily into thrombin Thrombin has a MW-33700, nearly half of the parent compound. Vitamin K is required by liver for prothrombin synthesis.

24. CONVERSION OF FIBRINOGEN TO FIBRIN-FORMATION OF THE CLOT

25. FIBRINOGEN HMW Protein, 340000 Plasma concentration, 100-700 mg/dl Liver disease can decrease the concentration of fibrinogen Only very little fibrinogen leaks from the blood vessels into the interstitial fluids and hence, interstitial fluids do not coagulate

26. ACTION OF THROMBIN ON FIBRINOGEN TO FORM FIBRIN Thrombin is a protein enzyme with weak proteolytic capabilities Form each molecule of fibrinogen 4 LMW peptides are removed-1 molecule of fibrin monomer is formed Fibrin monomer has capability to polymerize with other fibrin molecules to form fibrin fibers With in seconds, reticulum of the blood clot is formed with the long fibrin fibers Early stages, clot is weak and can be broken apart with ease

27. Contd… Then Fibrin Stabilizing Factor is released from platelets entrapped in the clot Same thrombin that causes fibrin formation activates the Fibrin Stabilizing Factor, before FSF can have effect on fibrin fibers Activated FSF forms strong covalent bond b/w monomer of the fibrin and multiple cross linkage b/w adjacent fibrin fibers.

28. BLOOD CLOT The clot is a meshwork Fibrin fibers also adhere to damaged surfaces of blood vessels.

29. CLOT RETRACTION-SERUM Contraction causes expression of fluid from clot-serum Platelet contractile proteins contributes greatly to the clot retraction by activating Platelet Thrombosthenin They also helps compress fibrin mesh work into smaller mass. As the clot contracts, the edges are further pulled together, contributing ultimate state of Hemostasis.

30. VISCIOUS CIRCLE OF CLOT FORMATION Positive feed back for clot formation Most important cause of this is the the proteolytic action of thrombin For instance, thrombin has a direct proteolytic effect on prothrombin itself, hence more of thrombin formation Critical amount of thrombin causes more blood clotting and hence further more production of thrombin and hence called a vicious circle of clotting.

31. INITIATION OF COAGUALTION FORMATION OF PROTHROMBIN ACTIVATOR These mechanisms are set into play by:- Trauma to the vascular wall and the adjacent tissues Contact of the blood with damaged endothelial cells Prothrombin activator is generally considered to be formed in these ways- a) Extrinsic pathway for initiating blood clotting b) Intrinsic pathway for initiating blood clotting

32. CLOTTING FACTORS Factor I (fibrinogen)- Fibrinogen is a soluble plasma protein (MW 330000) which is acted upon by thrombin to form insoluble fibrin clot. Factor II (prothrombin)- Inactive precursor of thrombin is formed in liver. Factor III (tissue factor, tissue extract, thromboplastin)- This converts prothrombin in the presence of factors V, VII, and Xa, Calcium, and phospholipid.

33. CONTD.. Factor IV (calcium)- Factor V (labile factor, thrombogene or proaccelerin )- This factor is reqd. for the conversion of prothrombin to thrombin by tissue extract and plasma factors. Factor VII (stable factor, autoprothrombin I)- Factor VII is reqd. for the formation of prothrombin activator by tissue extract. Factor VIII ( antihemophilic globulin [AHG], antihemophilic factor)- Factor VIII is reqd. for the formation of prothrombin activator from blood constituents; it ’ s consumed during clotting and hence absent from serum. In vivo the half life of factor VIII is 10-20h.

34. Factor IX (Christmas factor, plasma thromboplastin component, autoprothrombin II)- It ’ s needed for the formation of prothrombin activator from blood constituents.. Factor X (stuart-prower factor)- Converted to factor Xa either by factors IXa & VIII or factor VII & tissue factor. Factor X can be activated by other proteases such as trypsin or Russel ’ s viper venom. Factor XI (plasma thromboplastin acntecedent-PTA)- Also a beta 2 globulin present both in serum & alumina treated plasma. It ’ s thermolabile. Unlike factor XII, its activity increases when stored frozen. CONTD..

35. Factor XII ( Hageman factor or contact factor)- It takes part in the formation of prothrombin activator from blood constituents. It ’ s present in both serum & plasma. Factor XIII (fibrin stabilizing factor)- This is plasma protein which causes polymerization of soluble fibrin to produce insoluble fibrin. Fletcher factor- Described by Hathway (1965). Deficiency resembles factor XII deficiency. It ’ s a prekallikerin. Evidence indicating that prekallikerin is activated by limited proteolysis. Fitzgerald factor- Its heat stable. This appears to act after the activation of Hageman factor & Fletcher factor but before the activation of factor XI. It ’ s necessary for conversion of factor XI by Kaolin activated factor XII. It ’ s reqd. for normal fibrinolysis & kinin formation. CONTD..

36. FACTOR TRIVIAL NAMES PATHWAY PREKALLIKERIN FLETCHER FACTOR INTRINSIC HIGH MOLECULAR WEIGHT KININOGEN (HMWK) CONTACT ACTIVATION COFACTOR; FITZERALD; FLAUJEAC WILLIAMS FACTOR INTRINSIC FACTOR I FIBRINOGEN BOTH FACTOR II PROTHRMBIN BOTH FACTOR III TISSUE FACTOR EXTRINSIC FACTOR IV CALCIUM BOTH PRIMARY CLOTTING FACTORS

37. FACTOR V PROACCELERIN, LABILE FACTOR, ACCELERATOR (Ac-) GLOBULIN, THROMBOGENE BOTH FACTOR VI (Va) ACCELERIN - FACTOR VII PROCONVERTIN, SERUM PROTHROMBIN CONVERSION ACCELERATOR (SPCA), COTHROMBOPLASTIN, AUTOPROTHROMBIN I EXTRINSIC FACTOR VIII ANTIHEMOPHILIC FACTOR A, ANTIHEMOPHILIC GLOBULIN (AHG), PLATELET CO-FACTOR 1 INTRINSIC FACTOR IX CHRISTMAS FACTOR, ANTIHEMOPHILIC FACTOR B, PLASMA THROMBOPLASTIN COMPONENT (PTC) INTRINSIC FACTOR X STUART-PROWER FACTOR, AUTOPROTHROMBIN Ic OR III, BOTH .CONTD….

38. FACTOR XI PLASMA THROMBOPLASTIN ANTECEDENT (PTA), ANTIHEMOFHILLIC C, INTRINSIC FACTOR XII HAGEMAN FACTOR, CONTACT FACTOR, ANTIHEMOPHILLIC D INTRINSIC FACTOR XIII PROTRANSGLUTAMINASE, FIBRIN STABILIZING FACTOR (FSF), FIBINOLIGASE, LAKI-LORAND FACTOR BOTH FACTOR XIV FLETCHER FACTOR INTRINSIC FACTOR XV FITZERALD FACTOR INTRINSIC FACTOR XVI PASSOVOY FACTOR INTRINSIC FACTOR XVII WILLIAMS FACTOR INTRINSIC FACTOR XVIII FLAUJEAC FACTOR INTRINSIC CONTD….

39. ZYMOGENS OF SERENE PROTEASES ACTIVITIES FACTOR XII Binds to exposed collagen site of vessel wall injury, activated by HMW Kininogen & Kallikrein FACTOR XI Activated by factor XIIa FACTOR IX Activated by XIa in presence of Ca 2+ FACTOR VII Activated by thrombin in presence of Ca 2+ FACTOR X Activated on surface of activated platelets and by factor VIIa in presence of tissue factor and Ca 2+ FACTOR II Activated on surface of activated platelets by prothrombinase complex FUNCTIONAL CLASSIFIACATION OF CLOTTING MECHANISM

40. COFACTORS ACTIVITIES FACTOR VIII Activated by thrombin; factor VIIIa is a cofactor in the activation of factor X by factor IXa FACTOR V Activated by thrombin; factor Va is a cofactor in the activation of prothrombin by factor Xa FACTOR III (TISSUE FACTOR, EXTRINSIC THROMBOPLASTIN OR PLATELET FACTOR 3 ) A subendothelial cell-surface glycoprotein that acts as a cofactor for factor VII FIBRINOGEN ACTIVITY FACTOR I Cleaved by thrombin to form fibrin clot TRANSGLUTAMINASE ACTIVITY FACTOR XIII Activated by thrombin in presence of Ca 2+ ; stabilizes fibrin clot by covalent cross-linking CONTD….

41. REGULATORY & OTHER PROTEINS ACTIVITIES VON WILLIBRAND FACTOR Associated with subendothelial connective tissue; serves as a bridge between platelet glycoprotein GPlb /IX and collagen PROTEIN C Activated to protein Ca by thrombin bound to thrombomodulin; then degrades factors VIIIa and Va PROTEIN S Acts as a cofactor of protein C; both proteins contain gla residues THROMBOMODULIN Protein on the surface of endothelial cells; binds thrombin, which then activates protein C ANTITHROMBIN III Most important coagulation inhibitor, controls activities of thrombin, and factors IXa , XA, XIa and XIIa . CONTD….

42. EXTRINSIC PATHWAY FOR INITIATING BLOOD CLOTTING

43. EXTRINSIC PATHWAY The extrinsic pathway for initiating the formation of prothrombin activator begins with- a) traumatized vascular wall b) traumatized extravascular tissues which come in contact with the blood. This leads to following steps:- Release of tissue factor (TF) or tissue thromboplastin Activation of Factor X- role of Factor VII & TF Effect of Activated Factor X (Xa) to form Prothrombin activator- role of Factor V

44. INTRINSIC PATHWAY FOR INITIATING BLOOD CLOTTING INTRINSIC PATHWAY

45. 1. Trauma to blood vessel causes:- a) activation of Factor XII b) release of platelet phospholipids 2. Activation of Factor XI a) Factor XIIa acts on Factor XI to form Factor XIa b) this reaction also requires HMW kininogen and is accelerated by prekallikrein 3. Activation of Factor IX a) Factor XIa activates Factor IX to IXa 4. Activation of Factor X- role of Factor VIII a) Factor IXa acts in concert with Factor VIIIa & with the platelet phospholipids and Factor 3 from the traumatized platelets activate Factor X 5. Action of Factor Xa to form prothrombin activator- role of Factor V a) this step in intrinsic pathway is same as last step in extrinsic pahtway b ) Factor Xa + Factor V + tissue/phospholipids to form a complex called prothrombin activator The prothrombin activator in turns initiates with in seconds the cleavage of prothrombin to thrombin and hence, the final clotting process.

46. COMMON PATHWAY

47. ROLE OF CALCIUM IONS IN BOTH PATHWAYS Except for first two steps in the intrinsic pathway Ca 2+ ions are required for the promotion/acceleration of all the blood-clotting reactions In the living body Ca 2+ ion concentration seldom falls low enough to significantly affect the kinetics

48. INTERACTIONS B/W THE EXTRINSIC & INTRINSIC PATHWAYS-SUMMARY OF INITIATION

49. PREVENTION OF BLOOD CLOTTING IN THE NORMAL VASCULAR SYSTEM- INTRAVASCULAR ANTICOAGULANTS Endothelial surface factor a) smoothness of endothelial cells b) layer of Glycocalyx (mucopolysaccharide) c) Thrombomodulin d) Glycocalyx-thrombomodulin layer are lost from the endothelial wall there is activation of FXII and the platelets and hence intrinsic pathway e) If factor XII and the platelets come in direct contact with sub endothelial collagen the activation is even more powerful.

50. 2. ANTITHROMBIN ACTION OF FIBRIN & ANTITHROMBIN-III a) The fibrin fibers formed during the process of clotting adsorbs 85-90% of thrombin b) An alpha-globulin called antithrombin III or antithrombin-heparin cofactor 3. HEPARIN a) heparin when combines with antithrombin III, increases the effectiveness of the latter in removing thrombin b) this complex also removes activated Factors XII, XI, X, IX c) heparin is produced by basophillic mast cells in pericapillary CT. CONTD..

51. LYSIS OF BLOOD CLOTS Plasminogen or profibrinolysin when activated forms plasmin (fibrinolysin) Plasmin is a proteolytic enzyme that digests fibrin fibers and protein coagulants such as fibrinogen, Factor V, Factor VIII, prothrombin & Factor XII.

52. Streptokinase is an exogenous activator derived from beta-haemolytic streptococci FIBRINOLYTIC SYSTEM

53. ACTIVATION OF PLASMINOGEN TO FORM PLASMIN: THEN LYSIS OF CLOT Injured tissues and vascular endothelium gradually, after the clot has stopped the bleeding, release powerful activator, t-PA (tissue plasminogen activator) t-PA converts plasminogen to plasmin, which in turn removes the remaining unnecessary blood clot. Infact, many small blood vessels in which blood flow has been blocked are reopened by this mechanism.

54. Methods of haemostasis

55. Mechanical haemostasis

56. Direct pressure First choice to control bleeding Fast and simplest Small Arterial bleeding Venous bleeding 15-20 sec Not recommended in major artery and veins.

57. Fabric pads/gauze/sponge Used with direct pressure It is used in - only pressure is not an option -systemic bleeding due to infection, trauma, massive blood loss, and platelet dysfunction.

58. Suture/staples/ligating clips Suture – used in major arteries and veins Ligation of facial artery, lingual artery, and external carotid artery Stick Tie Ligation

59. Types of Ligation Stick Tie: Also called as transfixation . Used for High Blood pressure Proximal part of the vessels Regular Tie Used for Distal part of the vessels Also used for tubectomy . Regular Tie

60. Staples- sterile and disposable titanium staples Ligating clips- quick and easy decrease foreign body reaction various size

61. Use of Hemostats Hemostats (Mosquito and Artery) are designed to catch bleeders. Can be straight or curved. Various sizes –Micro mosquito,Hartman artery, Halstead mosquito, Crile ,Crile Ranklin , Kelly Ranklin,Long , Loop or sponge locking

62. Bone wax Is a mixture of Beeswax (70%) and Vaseline (30%). It is a non-absorbable material , becoming soft and malleable in the hand when warmed Its Hemostatic effect is based on physical rather than biochemical properties. It has been used in bone surgeries COMPLICATIONS:ALLERGIC, GRANULOMA, INFECTION, INTERFERES WITH BONE HEALING

63. Trans Catheter arterial embolization -Restricts tumors blood supply . -Arterial embolization preferentially interrupts tumors blood supply and stalls growth until neovascularization - Used to control bleeding in Hemangiomas

64. Thermal Energy Method Heat (Cautery) Electro cautery : it is the use of high frequency alternating current for cutting, coagulating, dessication or fulgurating tissue in both open and laparoscopic procedure monopolar electro surgery bipolar electro surgery bipolar electrosurgery vessel sealing technology argon enhanced coagulation technology Ultrasonic device Lasers

65. Monopolar electro surgery Most frequently used Two electodes- active (the pencil) - dispersive Modes - coagulation mode - cutting mode - blend mode Current flows through the patient from electrode (active) to electrode (dispersive)

66. Bipolar electro surgery Current does not flow through the patient’s body Lower voltage Indicated in limited thermal spread Delicate tissue, small anatomical tissue Safe for implanted medical devices such as pacemaker, internal cardioconverter fibrillator etc.

67. Bipolar electrosurgery sealing technology Advances electrosurgery modality in which the intimal layers of the vessel are fused and permanent seal is formed. Heat with compression Capable of simultaneously sealing and transecting vessel upto 7mm in diameter, large tissue pedicle and vascular bundles

68. Argon enhanced coagulation technology Used a stream of inert non combustible argon gas. Argon gas makes more conductive in electrosurgery Acts as a bridges between patientt and electrode In this monopolar current is transmitted to a tissue through the flow of argon gas .The tip of the coagulator is held 1 cm from the tissue . A flow of argon gas clears the surgical site of fluids to allow current to be focused directly on the tissue .

69. Ultrasonic device Converts electrical energy into mechanical energy Oscillate longitudinally at the point of contact, vibrating at 55,500/sec. Simultaneously cuts and coagulates Seal vessel upto 5 mm diameter Limits thermal damage to surrounding tissue No current through the body

70. Laser : Light amplification by stimulated emission of radiations. results in bloodless surgery. As they coagulate the small blood vessel during cutting of tissues. Cryosurgery :- Extreme cooling has been used for hemostasis .temperature ranging from -20 to -180 are used. Tissue capillaries ,small arterioles undergo cryogenic necrosis . This is caused by dehydration and denaturation of lipid molecules

71. Chemical methods Pharmacological agents Topical haemostatic agent Passive active

72. Pharmacological agents Sterile haemocoagulase solution Epinephrine Vitamin k Protamine Desmopressin Lysin analogs Etamsylate

73. Sterile haemocoagulase Eg . Botro clot, Reptilase inj. Contents – Haemocoagulaes - isolated from bothrox atrox Chlorhexidine solution Water Topical application of 5-10 drops , 1ml IM

74. Styptics Monsels solution – ferric subsulphate : Acts by precipitating protein. Used for capillary bleeding and post-extraction bleeding Tannic acid : Acts by precipitating proteins Home remedy for an emergency Mann Hemostatic – tannic acid , alum and chlorobutanol Others: -Silver nitrate -Ferric chloride

75. Epinephrine Causes direct vasoconstriction Can be applied topically and can be injected with LA Prolong analgesic effect Reduces bleeding during surgery Topical - The drug is applied with the help of gauze pack in concentration of 1:1000 over a oozing It is also injected along with local anesthetics in concentration of 1:80,000 and 1:2,00,000.

76. Vitamin K Plays important role in coagulation process Helps in production of fibrinogen and prothrombin in liver Route- orally and IV(slow) IM and subcutaneous is not recommended because irratic absorption Dose- Males: 120 mcg/day PO Females: 90 mcg/day PO  5-10 mg IV (dilute in 50 mL IV fluid and infuse over 20 min

77. Protamine Reverse heparin anticoagulation activity Adverse effect- anaphylaxis, acute pulmonary vasoconstriction, right ventricular failure Contraindication -diabetic -pt undergone vasectomy -drug allergy -previous protamine exposure Dose -1.0 -to- 1.5 mg protamine sulfate IV for every 100 IU of active heparin

78. Desmopressin Synthetic vasopressin analogues Stimulates the release of von willibrand factor and factor Viii from the endothelial cells. Enhances primary haemostasis Slow iv infusion Dose- 0.3 µg/kg diluted in 50ml saline and infused i.v over 30 min. Reduces perioperative bleeding

79. Lysine Analogues Eg . Epsilon Aminocaproic acid, tranexamic acid Inhibits the activation of plasminogen Reduces the conversion of plasminogen into plasmin Dose iv- Epsilon aminocaproic acid- 1-15gm(loading dose) followed by maintenance dose of 1-2gm hourly Total dose of 10-30gm Oral- 500mg , Inj – 5g/20 ml

80. Tranexamic acid- loading dose 2-7gm Follwed by 20-250 mg hourly Total dose of 3-10gm Oral dose; 500 mg 6-8 hrly Children; 1.25g/5 ml of syrup Inj- 0.5-1g slow i.v infusion TID

81. Ethamsylate Reduces capillary bleeding Increase the ability of platelets to stick together Dose; 250-500 mg 8 hourly Contraindication- hypersensitivity and blood porphyria.

82. Adenochrome Monosemicarbazone - Doubtful efficacy 1mg/ml inj is given as 2ml/6 hourly before surgery -Acts by correcting abnormal platelet adhesion Contraindications -Allergy to ingredients -Pregnancy & Lactation