May 9, 2017 ÒThe normal physiological response that prevents significant blood loss following vascular injury is called haemostasis.6 Familiarity with haemostasis lays the groundwork for a thorough understanding of the major disease states associated with thrombosis, such as venous thromboembolism (VTE), atherothrombosis (thrombosis triggered by plaque rupture), and cardioembolic stroke. Subendothelial matrix Platelets Hemostatic plug Fibrin Endothelial cell RBC WBC WBC Hemostasis refers to the prevention of blood loss, and is accomplished by vasoconstriction and coagulation by cellular and coagulation factors. Undue bleeding is controlled and the fluidity of the blood is maintained by counterbalances within the coagulation and fibrinolytic systems. Blood vessel injury or disruption, platelet defects, abnormalities of the normally circulating anticoagulants and fibrinolytic mechanisms may upset the balance between fibrinolysis and coagulation. Blood normally circulates through endothelium-lined vessels without coagulation or platelet activation occurring and without appreciable hemorrhage. Injury to the endothelial cells triggers the hemostatic process, which typically begins with the attachment of platelets (“Adhesion”) to the damaged endothelium or exposed subendothelial proteins such as collagen and von Willebrand factor (vWf). The platelets then change form (“Activate”) and release factors that stimulate the clotting process. They also bind together (“Aggregate”). At the same time, plasma proteins may react with elements in the subendothelium, activating the “contact” phase of coagulation. Exposed fibroblasts and macrophages present tissue factor, a membrane protein, to the blood at the injured site, thereby triggering the “Extrinsic “phase of blood coagulation. Under normal conditions, hemostasis protects the individual from massive bleeding secondary to trauma. In abnormal states, life-threatening bleeding can occur or thrombosis can occlude the vascular tree. Hemostasis is influenced by a number of different factors including: (a) vascular extracellular matrix and alterations in endothelial reactivity, (b) platelets, (c) coagulation proteins, (d) inhibitors of coagulation, and (e) fibrinolysis. Cotran RS, Kumar V, Robbins SL, eds. Robbins pathologic basis of disease, 5th ed. Philadelphia: W.B. Saunders, 1994 pp 99-106. Goodnight S. Physiology of coagulation and the role of vitamin K. In: Ansell JE, Oertel LB, Wittkowsky AK, eds. Managing oral anticoagulation therapy, Gaithersburg: Aspen Publishers, 1997 pp 1B-1:1-5. Fibrin Red Blood Cell Platelet This scanning electron photomicrograph shows the actual clot formation. The fibrin "mesh" of cross-linked fibrin monomers can be seen as a white stringlike substance trapping red blood cells in a fresh clot.The red cells are not sticking together; they are being held together by fibrin. Much the same process occurs early in clot development, when platelet aggregates are held together by fibrinogen, which stabilizes the first hemostatic plug. Colman RW, Hirsh J, Marder VJ, Salzman EW. Overview of hemostasis. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and thrombosis, 3rd ed. Philadelphia: J.B. Lippincott, 1994 pp 6,13,14. ÒExcessive coagulation leads to the formation of a thrombus, potentially obstructing blood flow. This is a common problem, especially in hospitalised or immobilised patients. Venous thromboembolic disease, for example, is a major problem in the European Union, where it causes more than one million events or deaths every year. Excessive bleeding results when certain coagulation factors are lacking, as in patients with haemophilia. ÒBlood vessel injury triggers the following sequence: ÒThe vessel constricts to reduce blood flow ÒCirculating platelets adhere to the vessel wall at the site of trauma ÒPlatelet activation and aggregation, coupled with an intricate series of enzymatic reactions involving coagulation proteins, produces fibrin to form a stable haemostatic plug Ò This finely tuned process serves to maintain the integrity of the circulatory system.However, the process can go out of balance, leading to significant morbidity and mortality. •Adhesion • •GpIIb/IIIa PLATELET ACTIVATION PATHWAYS (1) •GpIIb/IIIa •GpIIb/IIIa •Aggregation •ADP •Adrenaline •Platelet • • • • • • • • • • •Exposed Collagen •Endothelium • •vWF •COLLAGEN • • • • • •GpIIb/IIIa •GpIIb/IIIa •Aggregation •GpIIb/IIIa •GpIIb/IIIa •Aggregation •Adhesion • •Adhesion •ADP •Adrenaline • • THROMBIN Multiple pathways are responsible for platelet activation. Platelets adhere to damaged blood vessels via cell surface adhesion molecules and their membrane receptors such as glycoprotein Ib/IX (GP Ib/IX), the ligand for von Willebrand factor (VWF), which in turn can activated platelets and cause conformational changes. Further, other activators including thrombin, adrenaline, ADP, and collagen can also activate platelets. When activation occurs, the glycoprotein IIb/IIIa membrane receptor (GP IIb/IIIa) is exposed. This receptor forms bridges using fibrinogen resulting in aggregation. Platelet activation also exposes a phospholipid surface (meeting place) upon which coagulation proteins carry out their reactions. The sequential activation of these coagulation factors ultimately leads to the formation of fibrin, which is a critical component in stabilizing the hemostatic plug. Thrombin when generated, plays a pivotal role in hemostasis, via both fibrin conversion and platelet activation. • • Platelet Activation Pathways (2) • • Platelet Aggregation Fibrinogen • Fibrinogen Binding Site • • • Thrombin Platelet Herbert. Exp Opin Invest Drugs 1994;3:449-455. Ò ÒCoagulation involves a complex set of protease reactions involving roughly 30 different proteins. ÒThe final result of these reactions is to convert fibrinogen, a soluble protein, to insoluble strands of fibrin. Together with platelets, the fibrin strands form a stable blood clot. fibrinolysis-clot-formation-PU •Thrombogenic and antithrombogenic components in the body • An external file that holds a picture, illustration, etc. Object name is IJA-58-515-g001.jpg An external file that holds a picture, illustration, etc. Object name is IJA-58-515-g005.jpg •Palta S et al., Indian J Anaesth, 58:515-523, 2014 ÒThis model identifies membranes of cell presenting tissue factor )TF) and a surface of platelets as places of activation of specific coagulation factors. ÒThe model supposes the model of zhree phases: initiation, amplificatopn (propagation) and the proper action of thrombin- thrombus formation. ÒInitiation = formation of complex TF-FVIIa which is leading to avtivation of a small amount of thrombin. ÒPropagation = activation of platelets by thrombin and formation of complex FIXa-FVIIIa with subsequent activation of factor Xa. ÒThrombus formation = formation of prothrombinase complex anf of large amount of thrombin which is leading to formation of thrombus. Hemostasis-2011-6 C:\Users\prof. Vasku\Pictures\2015-04-15 Iniciace koagulace\Iniciace koagulace 001.jpg •Palta S et al., Indian J Anaesth, 58:515-523, 2014 ÒCoagulation cascade is activated when defect of vessel wall enables contact of the blood with cells with TF. ÒPlatelets membrane bound tissue factor TF activates FVII to VIIa which is leding to formation of complex TF-VIIa. ÒThe complex binding on platelets membranes activates Factor IX(a) and Factor X(a). ÒFactor Xa converts small amount of prothrombin (Faktor II) on trombin (Factor IIa) which can activate Factor V on FVa and Factor VIII on FVIIIa. C:\Users\prof. Vasku\Pictures\2015-04-15 Propagace koagulace\Propagace koagulace 001.jpg •Palta S et al., Indian J Anaesth, 58:515-523, 2014 Ò ÒFactor Xa together with activated Factor V (Va) as cofactor support coagulation by thrombin formation (Factor IIa ) from prothrombin (Factor II). ÒFactor Xa is primary pount for propagation of the porcess; one molecule of Factor Xa catalyses formation of about 1,000 molecules of thrombin. Ò ÒIn the final step, sequence of serin proteinases reactions which lead to formation of blood clot, thrombin will convert soluble fibrinogen to insoluble fibrin. ÒThrombin also activates Factor XIII (stabilizing fibrin) which can stabilize clot by crosslinking of fibrin. ÒStabilized fibrin is able to retain cellular components (red blood cells, platelets or both). S25798-08-f30 •Fibrinolýza C:\Users\prof. Vasku\Pictures\2015-04-15 Fibrinolýza\Fibrinolýza 001.jpg •Palta S et al., Indian J Anaesth, 58:515-523, 2014 Ò„Tissue factor plasminogen inhibitor“ –produced by endothelial cellls. It inhibits complex TF-VIIa. ÒAntithrombin (previously AT III) – binds activated vitamin K dependent coagulation factors (can be activated by heparin which increases its binding capacity) Ò„Protein Z dependent protease inhibitor/ protein Z (PZI)“ produced by liver. It inhibits FXa in presence PZ and Ca++. Ò Ò Ò Ò Ò Ò Ò Ò Ò Hemostasis-2011-3 Hemostasis 025 Hemostasis 024 Hemostasis 028 ÒNon activated-after their synthesis in liver ÒPosttranslationally modified –vitamin K dependent coagulation factors = serin proteázy ÒActivated –activated serin proteases, other activated factors (Va, VIIIa) Hemostasis 014 ÒScreening tests ÒBleeding time ÒPlatelet count ÒProthombin time (PT) ÒPartial thromboplastin time (PTT) ÒThrombin time (TT) ÒMore specific tests ÒVenous blood ÒExcessive stress and exercise cause changes in blood clotting. and fibrinolysis. ÒWhenever possible, venous samples should be collected without a pressure cuff (to avoid haemoconcentration, increase of fibrinolysis, platelet release, and activation of some clotting factors. ÒTo minimize the effect of contact activation plastic or polypropylene, siliconized glass, syringes and containers should be used. ÒThoroughly mixing the blood with the anticoagulant by inverting the containers several time. ÒThe sample should be brought to the laboratory as soon as possible. ÒLabeling the patient sample is very important. ÒAnticoagulant trisodium citrate 3.2 % in a ratio of 1 : 9. ÒTime of sample collection is very important factor in the interpretation of results. ÒCentrifugation and preparation of platelets poor plasma - 4000 rpm in a cooling centrifuge. ÉP.T & Factor VII à kept at room temperature. ÉOther assays à at 4oC. É ÉTesting should preferably be completed within 2 hours of the collection. ÒTime taken for bleeding to cease from a small superficial wound Ò ÒAffected by -Platelet count and function -Vessel wall - -Normal range Ivy’s method: 2-7 min ÒNormal platelet count = 150-400x109lL Ò ÒA part of complete blood picture (CBC) Ò ÒPerformed by electronic counters or manually (inherent error) ÒIndicates the overall efficiency of extrinsic pathway of blood coagulation (FVII, FII, FV, X) Ò ÒNormal range: 10-14 sec Ò ÒCauses of prolonged PT -Liver disease -Vit K deficiency (FII, V, VII, IX are Vit k dependent) -Deficiency of factors involved in extrinsic pathway -DIC -Oral anticoagulants Ò ÒIndicates the overall efficiency of intrinsic pathway of blood coagulation (FVIII, FIX, FXI, FXII, FII, FV, X) Ò ÒNormal range: 30-40 sec Ò Ò ÒCauses of prolonged PTT -Deficiency of factors involved in intrinsic pathway (coagulation factors other than FVII) -Liver disease -DIC -Massive transfusion (labile FV, FVIII) -Heparin Ò ÒProlonged PT + normal PTT= extrinsic pathway defect Ò ÒProlonged PTT + normal PT= intrinsic pathway defect Ò ÒProlonged PT and PTT= common pathway defect or combined factor deficiencies Ò ÒPlatelet count below 150x109/L ÒCauses: Ò- Congenital -Acquired - failure of production Ò Increased destruction (ITP) Ò- Splenic sequestration (hypersplenism) ÒITP is immune thrombocytopenia due to formation of antibodies against platelets and BM megakaryocytes. Ò ÒClinical picture: spontaneous bleeding purpuric eruptions. Ò ÒBT: prolonged ÒPlatelet count: thrombocytopenia ÒPT,PTT: normal ÒBM: increased megakaryocytes with poor platelet separation Ò ÒPlatelet function defect + normal plt count ÒCauses: -Hereditary (Glanzmann’s disease, Bernard-Soulier syndrome) -Acquired (drugs as aspirin, uremia) ÒClinical picture: spontaneous bleeding purpuric eruptions. Ò ÒBT: prolonged ÒPlatelet count: normal or slightly decreased ÒPT,PTT, TT: normal ÒPlatelet function: abnormal depending on the defect (defective aggregation in Glanzmann’s disease and Bernard-Soulier syndrome) Ò ÒHereditary thrombofilia ÒAT deficiency ÒProtein C deficiency ÒProtein S deficiency ÒFactor V Leiden ÒProthrombin polymorphism (G/A 20210 in 3´ area of the gene) ÒSy of antiphospholipid antibodies ÒIncreased levels of factors VIII, IX, XI and fibrinogen ÒFibrinolysis defects Ò Hemostasis 018 Hemostasis 029 nLMWH •Bleeding •Thrombocytopenia •Hypersensitivity ÒHeparin ÉBleeding ÉThrombocytopenia ÉOsteoporosis ÉHypersensitivity Bleeding still remains the most common side effect associated with heparin and LMWH. There has been reported a lower incidence of heparin-induced thrombocytopenia with LMWH. These findings may be due to the fact that LMWHs cause less activation of platelets and release of platelet factor 4 which yields fewer complexes. LMWH products should not be given to patients with HIT because there is a chance of cross-reactivity. When given for more than one month, unfractionated heparin can cause osteoporosis. The incidence of osteoporosis may be lower in patients given LMWH vs UFH. Weitz, Jeffrey I. Low-molecular-weight heparins. N Engl J Med.1997;337:688-698. Heparin (Heparin Sodium Injection, USP), Prescribing Information (1996), Eli Lilly & Co. Lovenox^® (Enoxaparin Sodium Injection, USP), Prescribing Information (1997), Rhone-Poulenc Rorer. Fragmin^® (Dalteparin Sodium Injection, USP), Prescribing Information (1997), Pharmacia & UpJohn Company. Orgaran^® (Danaparoid Sodium Injection, USP), Prescribing Information (1998), Organon, Inc. ÒFatal or non-fatal hemorrhage from any tissue or organ ÒNecrosis of skin and other tissues ÒOther adverse reactions reported less frequently include: ÉSystemic cholesterol microembolization ÉAlopecia ÉPurple toes syndrome, urticaria, dermatitis including bullous eruptions The adverse effects associated with warfarin are:  Fatal or non-fatal hemorrhage from any tissue or organ  Necrosis of skin and other tissues  Other adverse reactions reported less frequently include:  Body as a whole—pain, edema, asthenia, hypersensitivity/allergic reactions, fever, headache, malaise  Central and peripheral nervous system—dizziness, paresthesia  Gastrointestinal—nausea, diarrhea, abdominal pain, vomiting  Liver and biliary—elevated liver enzymes, hepatitis, jaundice, cholestatic hepatic injury  Platelet, bleeding, and clotting—systemic cholesterol microembolization  Skin and appendages—alopecia, rash pruritus, purple toes syndrome, urticaria, dermatitis including bullous eruptions  Vascular, extracardiac—vasculitis Coumadin® (Warfarin Sodium Tablets, USP) Crystalline, Prescribing Information (1998), DuPont Pharmaceuticals Company. Hemostasis 019 Hemostasis 021 Hemostasis 020 Hemostasis 022 Hemostasis 023 Hemostasis 027 Hemostasis 029 Hemostasis 019 ÒAF is the most common arrhythmia seen in clinical practice. ÒWithout appropriate anticoagulant treatment, most patients with AF are at increased risk of cardioembolic stroke. ÒCardiovascular disease is the leading cause of death in industrialised countries. Coronary artery disease (CAD) is the most common form of cardiovascular disease. In CAD, atherosclerosis damages the coronary artery wall, predisposing to thrombus formation. The symptoms and severity of acute coronary syndromes (unstable angina and myocardial infarction) vary depending on the degree to which thrombi occlude the coronary arteries. ÒPattern of bleeding: purpura Ò ÒCauses…… - -Screening tests for hemostasis: -BT: prolonged -Platelet count: normal -- PT, PTT, TT: normal Ò S25798-08-t22 S25798-08-t24 ÒAbnormal bleeding may result from -Vascular disorders -Thrombocytopenia ( ¯ platelet count) -Defective platelet function (qualitative defect) -Coagulation disorders ÒVon Willebrand´s disease ÒHemophilia A ÒHemophilia B ÒHemophilia C ÒFactor V deficiency ÒFactor VII deficiency ÒFactor XIII deficiency ÒProthrombin deficiency ÒAfibrinogenemia ÒConsumption coagulopathies ÒDIC-diseminated intravascular coagulation ÒMicroangiopathic hemolytic anemia ÒVitamin K deficinecy ÒLiver diseases Hemostasis 001 Hemostasis 002 ÒX-linked disorder ÒQuantitative or qualitative disorder of factor VIII ÒScreening tests: ÒBT: normal ÒPlatelet count: normal ÒPT: normal ÒPTT: prolonged ÒPlatelet count: normal ÒSpecific test: FVIII assay: decreased activity Ò S25798-08-f31 •(a) Factor VIII synthesis. •(b) Hemofilia A has a defect synthesis of VIIIc. •(c) von Willebrand ´s disease has a reducted synthesis of vWF ÒAlso called Chritmas disease ÒCompared to hemophilia A: -Less common -same presentation -Same screening tests results -Specific test: FIX assay: decreased activity Hemostasis 003 Hemostasis 004 ÒAutosomal dominant disease ÒQuantitative or qualitative disorder of vWF ÒVon Willebrand factor acts as a carrier for FVIII ÒActs as an essential cofactor for platelet adhesion and aggregation ÒScreening tests: ÒBT : prolonged. ÒPlatelet count: normal ÒPT: normal ÒPTT: prolonged Ò ÒSpecific tests: ÒPlatelet aggregation: defective with ristocetin ÒFVIII assay: decreased activity ÒvWF antigen : reduced Ò Ò Hemostasis 013 ÒRelease of tissue factor, TF. ÒTF is expressed on many cell types (endothelial, macrophages, monocytes). ÒContact with blood after damage of vessel wall (the effects of cytokines and endotoxins). ÒTF is binding to coagulation factors which is leading to activation of both pathways of coagulation cascades. Ò ÒDue to extensive coagulation followed by fibrinolysis with consumption of hemostatic factors. Ò ÒCauses: Òinfection, malignancy, obstetric complications, liver disease Ò ÒBT: prolonged ÒPlatelet count: decreased ÒPT: prolonged ÒPTT: prolonged ÒTT: prolonged ÒFibrinogen level: reduced ÒFDPs (D dimer): increased ÒRed cell fragmentation in the blood film BT PT PTT Platelet count Platelet function Other tests ITP P N N Glanzman P N N N Defect aggreg Hemoph A N N P N FVIII assay Hemoph B N N P N FIX assay vWD P N P N Defect aggreg FVIII, vWF DIC P P P Fibrinogen FDPs cat67