1 Rafi Ahmed, MD Hematology Oncology FellowHemostasis Rafi Ahmed, MD Hematology Oncology Fellow

2 Hemostasis Process of blood clot formation at site of vessel injuryResponse must be Quick Localized Carefully regulated

3 Phases of hemostasis Formation of platelet plugPropagation of clotting process by coagulation cascade Termination of clotting by antithombotic control mechanisms Removal of clot by fibrinolysis

4 Platelets Production Produced in Bone Marrow by fragmentation of cytoplasm of megakaryocytes Differentiation of stem cell to production of platelets is approximately 10 days Thrombopoetin is regulator of platelet production which binds to C-MPL receptors on platelets Lifespan is 7-10 days

5 Platelet Structure Glycoproteins of the surface involved in platelet reactions of adhesion and aggregation Interior with calcium, ADP, ATP, serotonin in granules. Alpha granules with heparin antagonist, PDGF, Beta-thromboglobulin, fibrinogen, VWF, and clotting factors

6 Platelet Function Formation of mechanical plugs during vascular injuryAdhesion Aggregation Secretion Procoagulant activity

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8 The Coagulation Cascade Primary HemostasisConsists of: Platelet plug formations Vascular spasm Capillary endothelial adhesions Temporary Fix Lasts hours

9 Primary Hemostasis Platelets adhere to exposed endothelial tissueSubendothelial microfibrils bind to subendothelial VWF (released from endothelium) This binds to GPIb-IX complex on platelet membrane Platelets now “roll” along surface until GPIa/IIa binds to collagen Platelet activation achieved by GPIIb/IIIa exposure to fibrinogen (primary) as well as VWF leading to platelet aggregation Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

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11 GP IB/IIA GP IIB/IIIA Collagen Receptor

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13 Primary Hemostasis Platelets adhere to exposed endothelial tissueSubendothelial microfibrils bind to subendothelialVWF (released from endothelium) This binds to GPIb-IX complex on platelet membrane Platelets now “roll” along surface until GPIa/IIa binds to collagen Platelet activation achieved by GPIIb/IIIa exposure to fibrinogen (primary) as well as VWF leading to platelet aggregation Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

14 Primary Hemostasis: Platelet release reaction and aggregationCollagen exposure/thrombin action: Secretion of platelet granules ADP, serotonin, fibrinogen, lysosomal enzymes, B thromboglobulin, and heparin neutralizing factors Activation of platelet prostaglandin synthesis Arachidonate release leads to thromboxane A2 Thromboxane A2 Potentiates platelet aggregation Vasoconstrictive activity Procoagulant Activity Phospholipid mediated reactions Factors IXa, VIIIa, and X in the formation of factor Xa Factors Xa, Va, and prothrombin II results in thrombin Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

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17 The Coagulation Cascade Secondary HemostasisSame time as platelets are aggregating Initiation substances sequentially activate by proteolysis a cascade of circulating precursor proteins Factors XII, XI, IX, VIII form the intrinsic pathway VIIa-Tissue Factor complex form the extrinsic pathway Both pathways leads to factor X activation Final pathway is converting prothrombin to thrombin which converts fibrinogen to fibrin Leads to firm and stable plugs

18 Secondary Hemostasis PTT PTImage retrieved from:

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21 Cofactor Cofactor Cofactor

22 Tissue Factor Cell bound glycoproteinPrincipal initiator of coagulation in vivo Mostly in extravascular location but gets exposed to blood when endothelial barrier is breached

23 Tissue Factor is a Co-FactorTF + VII TF-VIIA (active enzyme) X Xa (common pathway) IX IXa (intrinsic)

24 TFPI- Tissue Factor Pathway Inhibitor

25 Secondary Hemostasis In Vivo TF binds to factor VIIActivates factor VII (VIIa) VIIa-TF complex Activates factor IX and X Factor X leads to small amounts of thrombin Thrombin Activates cofactors V and VIII Amplification involves VIII and IX Continues to propagate factor Xa Activated factor X Converts prothrombin to thrombin Thrombin converts fibrinogen to fibrin monomer XIIIa stabilizes fibrin polymers Secondary Hemostasis Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

26 Cofactor Cofactor Cofactor

27 Coagulation Factor InhibitorsImportant effect of thrombin is limited to site of injury TFPI :tissue factor pathway inhibitor Protein C and S: inhibitors of coagulation cofactor V and VIII Thrombin binds to endothelial surface receptor thrombomodulin Activates vitamin K dependent serine protease protein C Protein S binds protein C to the platelet surface Anti Thrombin III

28 Protein C and Protein S ThrombomodulinCell bound Protein on Endothelium Binds to Thrombin (IIa) and inactivates Thrombomodulin/Thrombin Complex Protein C-> Activated Protein C (enzyme) Protein S is cofactor for APC that binds it to platelet surface Inactivates cofactors VIIIa and Va

29 Antithrombin III AntithrombinWeak Inhibitor of enzymes until in activated confirmation 1000X fold activated in presence of heparin Heparan sulfate proteoglycan on endothelial surface

30 Coagulation Factor InhibitorsFibrinolysis Generate Plasmin Remove Fibrin Fibrin Cofactor for destruction

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32 Fibrinolysis

33 Approach to Bleeding PatientAbnormal Bleeding May Result From Vascular disorders Thrombocytopenia Defective platelet function Defective coagulation

34 Pattern of Bleeding Vascular and Platelet DisordersAssociated with bleeding from mucous membranes and into skin Coagulation Disorders Bleeding in soft tissue or joints

35 Patient History Bleeding History Medication Use Past bleedingDuring surgical procedures Mucosal vs hemarthrosis Medication Use Aspirin, clopidogrel, ticlopidine, warfarin

36 Inherited Vascular DisordersHereditary hemorrhagic telangiectasia Autosomal dominant Dilated microvascular swellings Pulmonary AV malformations Recurrent GI hemorrhage Connective Tissue Disorders Ehlers-Danlos Hereditary collagen abnormalities Defective platelet aggregation

37 Acquired Vascular DefectsSenile purpura Atrophy of supporting tissues of cutaneous blood vessels on dorsal aspects of forearms and hands Purpura due to infections Vascular damage from immune complex formation Henoch-Schonlein In children, follows acute infection IgA mediated vasculitis Scurvy Vitamin C deficiency, defective collagen Steroid purpura Defective vascular support

38 Thrombocytopenia Production Defects Increased DestructionBone Marrow Failure Increased Destruction ITP HIT Drug induced TTP/HUS DIC Splenic Pooling

39 Hereditary Disorders of PlateletsThrombasthenia (Glanzmann’s Disease) Deficiency in glycoprotein IIb and IIIa Platelets fail to aggregate Bernard-Soulier Syndrome Platelets larger than normal Deficiency of glycoprotein Ib Defective binding to vWF, adherence, and no aggregation in ristocetin Variable thrombocytopenia Storage Pool Diseases

40 Acquired Platelet Functional DisordersLiver disease Decrease in clotting factors Quantitative and qualitative platelet disorders Cirrhosis: sequestration, decreased TPO Uremia Ecchymosis, epistaxis, GI/GU bleeding Cardiopulmonary Bypass Platelet interaction with non-physiologic surfaces Hypothermia, complement activation, cytokines, thrombin Adhesive properties altered

41 Von Willebrands DiseaseVWF Functions Primary Hemostasis Platelet Adhesion Platelet Aggregation Secondary Hemostasis Carrier of VIII in plasma (keeps it inactivated) until thrombin releases it Synthesized in endothelial cells and megakaryocytes Von Willebrand Disease Autosomal dominant

42 Von Willebrand DiseaseMucocutaneous bleeding usually mild to moderate Assays VWF activity: measures VWF binding to plts Ristocetin cofactor assay-gold standard Collagen binding, VWF:activity assay VWF antigen: concentration of protein (elisa) Factor VIII activity (carrier function of VWF) VWF Multimers, RIPA (Assays for classification) Type 1: Partial quantitative deficiency of VWF Type 2: (A, B, M, N) Qualitative defect in VWF Type 3: Total deficiency of VWF

43 Quick Review of Hemostasis Screening AssaysThrombin Time PT PTT Mixing Study

44 Thrombin Time Simple test to measure conversion of fibrinogen to fibrin Bovine thrombin added to citrated plasma, no calcium or phospholipid required (thus Lupus inhibitors don’t’ effect it) Prolonged by Heparin Hypo or dysfibrinogenemia High levels of FDP Circulating Paraprotein Antibody to bovine thrombin (cardiac patients) Reptilase time- will exclude Heparin Snake venom cleaves fibrinogen to fibrin Not affected by AT III

45 Prothrombin Time (PT) Thromboplastin (TF+PL+Ca2+) added to citrated plasma Trigger Extrinsic Pathway Usually insensitive to lupus inhibitors (since excess PL) Usually insensitive to heparin (adding heparinase to reagent) INR = (test PT/ mean normal PT) ^ISI (International sensitivity Index) Sensitivity to Deficiencies Factors VII, X, V, II (30-50 u/dl) (60-160) Fibrinogen mg/dL ( )

46 Activated Partial Thromboplastin Time APTTPartial Thromboplastin= Phospholipid + Calcium Activated: neg charged surface that activate contact factors (FXII, HMK) Added to citrated Plasma with Calcium Variable sensitivity to lupus inhibitors, heparin (less phospholipid)

47 Mixing Study 50-50 mix with normal plasma will correct if due to factor deficiency (50% of nl levels of any component factor) If not correct then concern for inhibitor Sometimes corrects but after incubation becomes prolonged- concern for inhibitor

48 Nl PT and prolonged PTT Disorders of intrinsic pathway DeficienciesFactor VIII: Hemophilia A, VWD Factor IX: Hemophilia B Factor XI (variable bleeding history) Factor XII, prekallikrein, HMWK (not associated with bleeding) Inhibitors Factor VIII (most common) Factor IX, XI (rare)

49 Prolonged PT and normal PTTDeficiency Factor VII deficiency (range of phenotype) Acquired inhibitors Factor VII rare Most commonly seen in warfarin therapy, early liver disease, vitamin K deficiency, DIC

50 Prolonged PT and PTT Deficiency of Factor X, V, II, ISuper therapeutic warfarin or heparin Vitamin K deficiency, liver disease, DIC, fibrinolysis

51 And finally some anticoagulantsVitamin K antagonists Warfarin (factor 10, 9, 7, 2, C, S) Activates Antithrombin III Heparin, LMWH Fondaparinux (pentasacchride) Direct Xa Inhibitors Rivaroxaban, apixaban Direct Thrombin Inhibitors Argatroban, Dabigatran