Pathophysiology of blood clotting disorders VLA 16.4.2020 Prof. Anna Vašků1 Hemostasis ̶ The 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. Prof. Anna Vašků2 Hemostasis Subendothelial matrix Platelets Hemostatic plug Fibrin Endothelial cell RBC WBC WBC Prof. Anna Vašků3 CLOT FORMATION Fibrin Red Blood Cell Platelet Prof. Anna Vašků4 Abnormal haemostasis ̶ Excessive 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. Prof. Anna Vašků5 Blood vessel injury ̶ Blood vessel injury triggers the following sequence: ̶ The vessel constricts to reduce blood flow ̶ Circulating platelets adhere to the vessel wall at the site of trauma ̶ Platelet activation and aggregation, coupled with an intricate series of enzymatic reactions involving coagulation proteins, produces fibrin to form a stable haemostatic plug ̶ 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. Prof. Anna Vašků6 Adhesion GpIIb/IIIa Platelet Activation Pathways (1) GpIIb/IIIaGpIIb/IIIa Aggregation ADP Adrenaline Platelet Exposed Collagen Endothelium vWF COLLAGEN GpIIb/IIIaGpIIb/IIIa AggregationGpIIb/IIIaGpIIb/IIIa Aggregation AdhesionAdhesion ADP Adrenaline THROMBIN Prof. Anna Vašků7 Platelet Activation Pathways (2) Platelet Aggregation Fibrinogen Fibrinogen Binding Site Thrombin Platele t Herbert. Exp Opin Invest Prof. Anna Vašků8 The coagulation cascade ̶ Coagulation involves a complex set of protease reactions involving roughly 30 different proteins. ̶ The 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. Prof. Anna Vašků9 Prof. Anna Vašků10 Prof. Anna Vašků11 Palta S et al., Indian J Anaesth, 58:515-523, 2014 Prof. Anna Vašků12 „Cell-based model“ ̶ This model identifies membranes of cell presenting tissue factor )TF) and a surface of platelets as places of activation of specific coagulation factors. ̶ The model supposes the model of three phases: initiation, amplification (propagation) and the proper action of thrombin- thrombus formation. ̶ Initiation = formation of complex TF-FVIIa which is leading to activation of a small amount of thrombin. ̶ Propagation = activation of platelets by thrombin and formation of complex FIXa-FVIIIa with subsequent activation of factor Xa. ̶ Thrombus formation = formation of prothrombinase complex anf of large amount of thrombin which is leading to formation of thrombus. Prof. Anna Vašků13 Prof. Anna Vašků14 Iniciation phase of coagulation Palta S et al., Indian J Anaesth, 58:515-523, 2014Prof. Anna Vašků15 Initiation phase of coagulation ̶ Coagulation cascade is activated when defect of vessel wall enables contact of the blood with cells with TF. ̶ Platelets membrane bound tissue factor TF activates FVII to VIIa which is leding to formation of complex TF-VIIa. ̶ The complex binding on platelets membranes activates Factor IX(a) and Factor X(a). ̶ Factor Xa converts small amount of prothrombin (Faktor II) on trombin (Factor IIa) which can activate Factor V on FVa and Factor VIII on FVIIIa. Prof. Anna Vašků16 Propagation phase of coagulation Palta S et al., Indian J Anaesth, 58:515-523, 2014Prof. Anna Vašků17 Propagation of coagulation: central role for Factor Xa ̶ Factor Xa together with activated Factor V (Va) as cofactor support coagulation by thrombin formation (Factor IIa ) from prothrombin (Factor II). ̶ Factor Xa is primary pount for propagation of the porcess; one molecule of Factor Xa catalyses formation of about 1,000 molecules of thrombin. Prof. Anna Vašků18 Final step: fibrin formation ̶ In the final step, sequence of serin proteinases reactions which lead to formation of blood clot, thrombin will convert soluble fibrinogen to insoluble fibrin. ̶ Thrombin also activates Factor XIII (stabilizing fibrin) which can stabilize clot by crosslinking of fibrin. ̶ Stabilized fibrin is able to retain cellular components (red blood cells, platelets or both). Prof. Anna Vašků19 Fibrinolýza Prof. Anna Vašků20 Palta S et al., Indian J Anaesth, 58:515-523, 2014Prof. Anna Vašků21 Natural inhibitors of coagulation ̶ „Tissue factor plasminogen inhibitor“ –produced by endothelial cellls. It inhibits complex TF-VIIa. ̶ Antithrombin (previously AT III) – binds activated vitamin K dependent coagulation factors (can be activated by heparin which increases its binding capacity) ̶ „Protein Z dependent protease inhibitor/ protein Z (PZI)“ produced by liver. It inhibits FXa in presence PZ and Ca++. Prof. Anna Vašků22 Prof. Anna Vašků23 Prof. Anna Vašků24 Prof. Anna Vašků25 Prof. Anna Vašků26 Coagulation factors -state ̶ Non activated-after their synthesis in liver ̶ Posttranslationally modified –vitamin K dependent coagulation factors = serin proteázy ̶ Activated –activated serin proteases, other activated factors (Va, VIIIa) Prof. Anna Vašků27 Prof. Anna Vašků28 Prof. Anna Vašků29 TESTS Screening tests Bleeding time Platelet count Prothombin time (PT) Partial thromboplastin time (PTT) Thrombin time (TT) More specific tests Prof. Anna Vašků30 Sampling ̶ Venous blood ̶ Excessive stress and exercise cause changes in blood clotting. and fibrinolysis. ̶ Whenever possible, venous samples should be collected without a pressure cuff (to avoid haemoconcentration, increase of fibrinolysis, platelet release, and activation of some clotting factors. ̶ To minimize the effect of contact activation plastic or polypropylene, siliconized glass, syringes and containers should be used. ̶ Thoroughly mixing the blood with the anticoagulant by inverting the containers several time. ̶ The sample should be brought to the laboratory as soon as possible. ̶ Labeling the patient sample is very important. Prof. Anna Vašků31 Sampling Anticoagulant trisodium citrate 3.2 % in a ratio of 1 : 9. Time of sample collection is very important factor in the interpretation of results. Centrifugation and preparation of platelets poor plasma - 4000 rpm in a cooling centrifuge. P.T & Factor VII → kept at room temperature. Other assays → at 4oC. Testing should preferably be completed within 2 hours of the collection. Prof. Anna Vašků32 BLEEDING TIME Time taken for bleeding to cease from a small superficial wound Affected by - Platelet count and function - Vessel wall - Normal range Ivy’s method: 2-7 min Prof. Anna Vašků33 PLATELET COUNT Normal platelet count = 150-400x109lL A part of complete blood picture (CBC) Performed by electronic counters or manually (inherent error) Prof. Anna Vašků34 PROTHROMBIN TIME Indicates the overall efficiency of extrinsic pathway of blood coagulation (FVII, FII, FV, X) Normal range: 10-14 sec Prof. Anna Vašků35 PROTHROMBIN TIME Causes 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 Prof. Anna Vašků36 PARTIAL THROMBOPLASTIN Indicates the overall efficiency of intrinsic pathway of blood coagulation (FVIII, FIX, FXI, FXII, FII, FV, X) Normal range: 30-40 sec Prof. Anna Vašků37 PARTIAL THROMBOPLASTIN Causes of prolonged PTT - Deficiency of factors involved in intrinsic pathway (coagulation factors other than FVII) - Liver disease - DIC - Massive transfusion (labile FV, FVIII) - Heparin Prof. Anna Vašků38 PT & PTT Prolonged PT + normal PTT= extrinsic pathway defect Prolonged PTT + normal PT= intrinsic pathway defect Prolonged PT and PTT= common pathway defect or combined factor deficiencies Prof. Anna Vašků39 THROMBOCYTOPENIA Platelet count below 150x109/L Causes: - Congenital - Acquired - failure of production  Increased destruction (ITP) - Splenic sequestration (hypersplenism) Prof. Anna Vašků40 Idiopathic Thombocytopenic Purpura ITP is immune thrombocytopenia due to formation of antibodies against platelets and BM megakaryocytes. Clinical picture: spontaneous bleeding purpuric eruptions. BT: prolonged Platelet count: thrombocytopenia PT,PTT: normal BM: increased megakaryocytes with poor platelet separation Prof. Anna Vašků41 QUALITATIVE PLATELET DEFECT Platelet function defect + normal plt count Causes: - Hereditary (Glanzmann’s disease, Bernard-Soulier syndrome) - Acquired (drugs as aspirin, uremia) Prof. Anna Vašků42 QUALITATIVE PLATELET DEFECT Clinical picture: spontaneous bleeding purpuric eruptions. BT: prolonged Platelet count: normal or slightly decreased PT,PTT, TT: normal Platelet function: abnormal depending on the defect (defective aggregation in Glanzmann’s disease and Bernard-Soulier syndrome) Prof. Anna Vašků43 Hereditary Thrombophilia ̶ Hereditary thrombofilia ̶ AT deficiency ̶ Protein C deficiency ̶ Protein S deficiency ̶ Factor V Leiden ̶ Prothrombin polymorphism (G/A 20210 in 3´ area of the gene) Prof. Anna Vašků44 Acquired Thrombotic disorders ̶ Sy of antiphospholipid antibodies ̶ Increased levels of factors VIII, IX, XI and fibrinogen ̶ Fibrinolysis defects Prof. Anna Vašků45 Prof. Anna Vašků46 Prof. Anna Vašků47 ◼ LMWH • Bleeding • Thrombocytopenia • Hypersensitivity Heparin/LMWH—Adverse Effects ̶ Heparin ̶ Bleeding ̶ Thrombocytopenia ̶ Osteoporosis ̶ Hypersensitivity Prof. Anna Vašků48 Warfarin—Adverse Effects ̶ Fatal or non-fatal hemorrhage from any tissue or organ ̶ Necrosis of skin and other tissues ̶ Other adverse reactions reported less frequently include: ̶ Systemic cholesterol microembolization ̶ Alopecia ̶ Purple toes syndrome, urticaria, dermatitis including bullous eruptions Prof. Anna Vašků49 Prof. Anna Vašků50 Prof. Anna Vašků51 Prof. Anna Vašků52 Prof. Anna Vašků53 Prof. Anna Vašků54 Prof. Anna Vašků55 Prof. Anna Vašků56 Prof. Anna Vašků57 Thrombosis and AF ̶ AF is the most common arrhythmia seen in clinical practice. ̶ Without appropriate anticoagulant treatment, most patients with AF are at increased risk of cardioembolic stroke. Prof. Anna Vašků58 Thrombosis and coronary artery disease ̶ Cardiovascular 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. Prof. Anna Vašků59 VASCULAR DISORDERS Pattern of bleeding: purpura Causes…… - Screening tests for hemostasis: - BT: prolonged - Platelet count: normal - - PT, PTT, TT: normal Prof. Anna Vašků60 Prof. Anna Vašků61 Prof. Anna Vašků62 BLEEDING DISORDERS Abnormal bleeding may result from - Vascular disorders - Thrombocytopenia (  platelet count) - Defective platelet function (qualitative defect) - Coagulation disorders Prof. Anna Vašků63 Hereditary Bleeding diseases ̶ Von Willebrand´s disease ̶ Hemophilia A ̶ Hemophilia B ̶ Hemophilia C ̶ Factor V deficiency ̶ Factor VII deficiency ̶ Factor XIII deficiency ̶ Prothrombin deficiency ̶ AfibrinogenemiaProf. Anna Vašků64 Acquired bleeding disorders ̶ Consumption coagulopathies ̶ DIC-diseminated intravascular coagulation ̶ Microangiopathic hemolytic anemia ̶ Vitamin K deficinecy ̶ Liver diseases Prof. Anna Vašků65 Prof. Anna Vašků66 Prof. Anna Vašků67 Hemophilia A X-linked disorder Quantitative or qualitative disorder of factor VIII Screening tests: BT: normal Platelet count: normal PT: normal PTT: prolonged Platelet count: normal Specific test: FVIII assay: decreased activity Prof. Anna Vašků68 (a) Factor VIII synthesis. (b) Hemofilia A has a defect synthesis of VIIIc. (c) von Willebrand ´s disease has a reducted synthesis of vWFProf. Anna Vašků69 Hemophilia B Also called Chritmas disease Compared to hemophilia A: - Less common - same presentation - Same screening tests results - Specific test: FIX assay: decreased activity Prof. Anna Vašků70 Prof. Anna Vašků71 Prof. Anna Vašků72 Von Willebrand Disease ̶ Autosomal dominant disease ̶ Quantitative or qualitative disorder of vWF ̶ Von Willebrand factor acts as a carrier for FVIII ̶ Acts as an essential cofactor for platelet adhesion and aggregation Prof. Anna Vašků73 Von Willebrand Disease Screening tests: BT : prolonged. Platelet count: normal PT: normal PTT: prolonged Specific tests: Platelet aggregation: defective with ristocetin FVIII assay: decreased activity vWF antigen : reduced Prof. Anna Vašků74 Prof. Anna Vašků75 DIC (disseminated intravascular coagulation) Release of tissue factor, TF. TF is expressed on many cell types (endothelial, macrophages, monocytes). Contact with blood after damage of vessel wall (the effects of cytokines and endotoxins). TF is binding to coagulation factors which is leading to activation of both pathways of coagulation cascades.  Prof. Anna Vašků76 Disseminated intravascular coagulation (DIC) Due to extensive coagulation followed by fibrinolysis with consumption of hemostatic factors. Causes: infection, malignancy, obstetric complications, liver disease Prof. Anna Vašků77 Diagnosis of DIC BT: prolonged Platelet count: decreased PT: prolonged PTT: prolonged TT: prolonged Fibrinogen level: reduced FDPs (D dimer): increased Red cell fragmentation in the blood film Prof. Anna Vašků78 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 Prof. Anna Vašků79 Thank you for your attention Prof. Anna Vašků80