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1 Functional Aspects of Slide 2 2 Homeostasis: State of fluid equilibrium within the blood vessels Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins Slide 3 3 Hemostasis Homeostasis Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins BleedingClotting Slide 4 4 Platelets Fibrinolysis/Inhibitors Coagulation Proteins Vessels Slide 5 5 Vascular System Endothelial Cells Basement Membrane Red Blood CellsPlateletsWhite Cells Slide 6 6 Platelets Fibrinolysis/Inhibitors Coagulation Proteins Vessels Slide 7 7 Disk-shaped cells produced in the megakaryocytes of the bone marrow Mature Platelet Megakaryocyte Bone Marrow What are Platelets? Slide 8 8 Anatomy of a Platelet Dense Body ADPSerotonin ATPCalcium Microtubular System Glycogen Exterior Coat (Glycocalyx) Mitochondria Surface Connecting System Cytosol Factor XIII Alpha Granules PF 4 Fibrinogen Factor VFibronectin Factor VIII R:ag Thromboglobulin (vWF) Plasma Membrane PF 3 Dense Tubular System Slide 9 9 Storage and Circulation Quantity - 200,000 - 400,000/mm 3 Life Span - 10 days 33% pooling 67% in the circulation Megakaryocyte Spleen Slide 10 10 Platelet Function Aggregation Coagulation Shape Change Release Fibrin Formation A B C 3 seconds 10 seconds 5 minutes ADP Release Adhesion Slide 11 11 Slide 12 12 Slide 13 13 Platelet Testing Peripheral Smear Platelet Count Platelet Aggregation Bleeding Time Slide 14 14 Platelet Aggregation Platelet Rich Plasma (PRP) Aggregating Reagent Aggregate Clumping Baseline Light Transmission Increased Light Transmission + Slide 15 15 Typical Biphasic Pattern 100 90 80 70 60 50 40 30 20 10 0 Platelet Rich Plasma Platelet Poor Plasma Secondary Response (Release) Primary Response Injection Point Lag Slide 16 16 Cut 1 mm deep 5 mm long Constant Pressure Expected Range 2 - 10 minutes 40 mm Bleeding Time Slide 17 17 Platelets Coagulation Proteins Fibrinolysis/Inhibitors Vessels Slide 18 18 Coagulation Factors Factor IFibrinogen Factor IIProthrombin Factor IIITissue Thromboplastin Factor IVCalcium Ions Factor VLabile Factor, Proaccelerin Factor VIIStable Factor, Proconvertin Factor VIIIAntihemophilic Factor Factor IXChristmas Factor Factor XStuart-Prower Factor Factor XIPlasma Thromboplastin Antecedent Factor XIIHageman Factor Factor XIIIFibrin Stabilizing Factor Slide 19 19 Fibrinogen VII Extrinsic Pathway (PT) Tissue Factor Kinins X X IX XI XII HMWK Prekalli- krein Kalli- krein XIIa XIa IXa VIIIa Xa VIIa Pro- Urokinase Intrinsic Pathway (aPTT) Fibrinolysis VIII Ure- Kinase T-PA Plas- minogen Plasmin Common Pathway IIVa V IIa XIII XIIIa Fibrin Polymer Fibrin Clot + Platelet Plug Slide 20 20 Coagulation Factors Fibrinogen Group Factors I, V, VIII and XIII Prothrombin Group Factors II, VII, IX and X Contact Group Factors XI, XII Prekallikrein (Fletcher Factor) High Molecular Weight Kininogen (Fitzgerald Factor) Slide 21 21 Fibrinogen Factor I Biosynthesis: Liver MW: 340,000 daltons Plasma Concentration: 2500 mg/L In Vivo Half-Life: 20 hours Pathology: Afibrinogenemia, autosomal recessive. Dysfibrinogenemia, autosomal dominant Factor IIa Factor IIa Fibrinopeptide A Fibrinopeptide B Fibrinogen Factor II Fibrin Monomer Procoagulant Activation Factor IIa Activation Procoagulant Fibrin Polymer CA+ Factor XIII Factor XIIIa Soluble Fibrin Polymer Plasmin Anticoagulant Inhibition Procoagulant Activation Fibrin (Factor Ia) Stable Thrombus (Clot) Activated Platelets FDPs Fibrinogen Degradation Products FDPs & XDPs (Cross-linked DPs) (e.g. D-dimers) Factor XIII Fibrin Stabilizing Factor (FSF) Biosynthesis: Megakaryocytes, liver MW: 320,000 daltons Plasma Concentration: 10mg/L In Vivo Half-Life: 12 days Pathology: FSF deficiency, autosomal recessive Slide 22 22 Factor V Proaccelerin, Labile Factor Biosynthesis: Liver, megakaryocytes MW: 330,000 daltons Plasma Concentration: 5-12 mg/L In Vivo Half-Life: 25 hours Pathology: Parahemophilia, autosomal recessive Slide 23 23 Factor VIII Antihemophilic Factor vWF von Willebrand Factor Biosynthesis: Liver, endothelium; Factor VIII Related Antigen, megakaryocyte MW(FVIII + vWF): 1.2-2 million daltons (6-10 subunits 200,000 daltons each) Plasma Concentration: 7 mg/L (vWF) In vivo Half-Life: 10 hours (Factor VIII) Pathology: Factor VIII-Hemophilia A, x-linked recessive. vWF-von Willebrands disease, autosomal dominant Factor IIa Factor VIII Activated Protein C Protein S Phospholipid (Platelet Factor 3) Inactive Fragments Ca++ Factor VIIIa Protein C Complex Activation Procoagulant Anticoagulant Inhibition Factor Xa or Slide 24 24 Factor II Prothrombin Biosynthesis: Liver, Vitamin K dependent MW: 70,000 daltons Plasma Concentration: 100 mg/L In Vivo Half-Life: 100 hours Pathology: Hypoprothrombinemia, autosomal recessive Factor II Factor IIa (Thrombin) Anticoagulant Inhibition Phospholipid (Platelet Factor 3) Activation Procoagulant Factor Va Factor Xa Ca++ * or Heparin Cofactor II Prothrombinase Complex Factor IIa Anti- thrombin III Heparin Anti- thrombin III Heparin * Activation Fragments Slide 25 25 Factor VII Proconvertin, Stable Factor Biosynthesis: Liver, Vitamin K dependent MW: 55,000 daltons Plasma Concentration: 1 mg/L In Vivo Half-Life: 5 hours Pathology: Hypoproconvertinemia, autosomal recessive Slide 26 26 Factor IX Christmas Factor Biosynthesis: Liver, Vitamin K dependent MW: 57,000 daltons Plasma Concentration: 4 mg/L In Vivo Half-Life: 20 hours Pathology: Hemophilia B, (Christmas disease) x-linked recessive Ca++ Tissue Factor VIIa Factor IXa Factor IX Factor IXa Activation Procoagulant Anticoagulant Inhibition Factor XIa or Anti- thrombin III Heparin Anti- thrombin III Heparin Slide 27 27 Factor X Stuart-Prower Factor Biosynthesis: Liver, Vitamin K dependent MW: 55,000 daltons Plasma Concentration: 5 mg/L In Vivo Half-Life: 65 hours Pathology: Stuart disease, autosomal recessive Tissue Factor Factor III, Thromboplastin Biosynthesis: Brain, lung, subendothelium MW: 45,000 daltons Factor VIIIa Tissue Factor Phospholipid (Platelet Factor 3) Factor IXa Factor VIIa Factor X Factor Xa Anti- thrombin III Heparin Anti- thrombin III Ca++ Heparin Activation Procoagulant Anticoagulant Inhibition or Extrinsic X-ase Complex Intrinsic X-ase Complex Slide 28 28 Factor XI Plasma Thromboplastin Antecedent Biosynthesis: Liver MW: 158,000 daltons Plasma Concentration: 4 mg/L In Vivo Half-Life: 65 hours Pathology: Hemophilia C, autosomal recessive Ca++ High Molecular Weight Kininogen Factor XIIa Factor XIa Factor XI Factor XIa Activation Procoagulant Anticoagulant Inhibition 1- Anti- trypsin 1- Anti- trypsin Slide 29 29 Prekallikrein Fletcher Factor Biosynthesis: Probably Liver MW: 107,000 daltons Plasma Concentration: 50 mg/L Pathology: Fletcher trait, autosomal recessive Ca++ High Molecular Weight Kininogen Factor XIIa Kallikrein Prekalli- krein Kalli- krein Activation Procoagulant Anticoagulant Inhibition C1 Esterase Inhibitor C1 Esterase Inhibitor Slide 30 30 Factor XII Hageman Factor Biosynthesis: Liver MW: 80,000 daltons Plasma Concentration: 29 mg/L In Vivo Half-Life: 60 hours Pathology: Hageman trait, autosomal recessive High Molecular Weight Kininogen (HMWK) Fitzgerald Factor MW: 120,000 daltons Plasma Concentration: 70 mg/L Pathology: Fitzgerald trait, autosomal recessive High Molecular Weight Kininogen Negatively Charged Activating Surface (e.g., kaolin, ellagic acid silica) Kalli- krein Factor XII Kinins High Blood Pressure and Inflammation Factor XIIa HMWK C1 Esterase Inhibitor C1 Esterase Inhibitor Factor XIIa * FXIIf alter vascular permeability Factor XII Fragments* Activation Procoagulant Anticoagulant Inhibition Slide 31 31 Extrinsic Pathway (PT) Common Pathway Fibrinigen VII X Xa VIIa II Va V IIa XIII XIIIa Fibrin Polymer Tissue Factor Fibrin Clot + Platelet Plug Slide 32 32 Prothrombin Time Thromboplastin and Calcium Patients Plasma Factors I II V VII X Slide 33 33 Fibrinogen Kinins X IX XI XII HMWK Prekalli- krein Kalli- krein XIIa XIa IXa VIIIa Xa Intrinsic Pathway (aPTT) VIII Common Pathway IIVa V IIa XIII XIIIa Fibrin Polymer Fibrin Clot + Platelet Plug Slide 34 34 Activated Partial Thromboplastin Time Ca++ Patients Plasma Factors I II V VIII IX X XI XII Phospholipid and Activator Slide 35 35 Quantitative Fibrinogen High concentration of thrombin 1:10 dilution patients plasma Time (in seconds) Fibrinogen in mg/dL 204060100200400600 60 30 10 6 3 1:40 1:30 1:20 1:10 1:5 Slide 36 36 Thrombin Clotting Time Low concentration of thrombin Undiluted patients plasma Screens for effects of Heparin FDPs Slide 37 37 Monitoring Anticoagulant Therapy Vitamin K Antagonists Antithrombin III Accelerators Slide 38 38 Vitamin K Antagonists Coumarin, Dicumarol, Warfarin Oral Administration Long-Acting Effect Factors IIa, VIIa, IXa, Xa PT Slide 39 39 AT-III Accelerators Heparin, Heparinoids, LMW Heparin Parenteral Administration Short-Acting Effect Factors IIa, IXa, Xa APTT Slide 40 40 Heparin/AT-III Complex Slide 41 41 Variables in Monitoring Heparin Time of Specimen Collection Type of Anticoagulant Centrifugation of Specimen Instrumentation and Reagents Slide 42 42 Factor Assays Diagnosis Therapy Slide 43 43 Factor Assays of the Extrinsic Proteins II, V, VII, X Plasma with 0% F.VII has a Prolonged Prothrombin Time By adding various concentrations of Factor VII Standard the Prothrombin Time is corrected The degree of correction is proportional to the concentration 0.1 mL F.VII Deficient Substrate 0.1 mL F.VII Standard or patients plasma dilution 0.2 mL Thromboplastin Calcium Chloride Mixture Slide 44 44 Factor Assays of the Intrinsic Proteins VIII, IX, XI, XII By adding various concentrations of Factor VIII Standard the APTT is shortened 0.1 mL Factor VIII Deficient Plasma Plasma with 0% Factor VIII has a prolonged APTT The degree of correction is proportional to the concentration 0.1 mL Factor VIII Standard or patients plasma dilution 0.1 mL Phospholipid and Activator 0.1 mL Calcium Chloride Slide 45 45 The Standard Curve Dilution% ActivityClotting Time 1:520.0%14.3 seconds 1:1010.0%17.3 seconds 1:205.0%21.0 seconds 1:402.5%26.0 seconds 30 25 20 15 10 5 2 5 10 20 Slide 46 46 Minimum Plasma Levels MinorMajor FactorSpontaneousTrauma Hemorrhageor Surgery I Fibrinogen50-100100 mg/dL II10-1520-40% V5-1525% VII5-1010-20% VIII5-1010-20% Hemophilia A15-2025% von Willebrand2525% IX10-1520-25% X5-1015-20% XI5-1515-25% XII1010% XIII15% Williams, W. J., Hematology, 1972 Slide 47 47 Platelets Fibrinolysis/Inhibitors Coagulation Proteins Vessels Slide 48 48 Fibrinolysis Activators Plasminogen Plasmin Fibrinogen Fibrin Degradation Products R.E.S. Slide 49 49 Plasminogen Biosynthesis: Liver MW: 90,000 daltons Plasma Concentration: 120 mg/L In Vivo Half-Life: 48 hours Pathology: Plasminogen deficiency, autosomal dominant. Dysplasminogenemia, autosomal recessive Urokinase Kallikrein tPA (tissue Plasminogen Activator) Plasminogen 2 - Anti- plasmin 2 - Anti- plasmin Activation Procoagulant Anticoagulant Inhibition Plasmin or Slide 50 50 Thrombolytic Therapy t-PA Streptokinase Urokinase Anaphylaxis GI Bleeding Intracranial Hemorrhage Slide 51 51 Antithrombin-III Inhibition of Thrombin Thrombin, Antithrombin-III and Heparin Heparin Lysine Site Acidic Site Arginine Site Serine Site Thrombin (Active) Antithrombin-III (Nonactivated) Inactivation of Thrombin Thrombin (Inhibited) Heparin Antithrombin-III Heparin Thrombin Complex Antithrombin-III (Complexed) Slide 52 52 Antithrombin-III Decreased Levels 1. Congenital 2. Acquired decreased synthesis 3. Acquired increased utilization 4. Drug-induced Slide 53 53 Protein C Vitamin K-dependent plasma protein Inactivates Factors V and VIII Stimulates fibrinolysis Slide 54 54 Protein C Biosynthesis: Liver, Vitamin K dependent MW: 56,000 daltons Plasma Concentration: 3-5 mg/L In Vivo Half-Life: 6-7 hours Pathology: Protein C deficiency, autosomal recessive (?) * Requires Protein S for functional activity Thrombo- modulin Factor IIa Protein C Activated Protein C Inhibitor Protein C Inhibitor Activated Protein C* Protein C Activation Peptide Activation Procoagulant Anticoagulant Inhibition Slide 55 55 Deficiencies of Protein C I. Congenital Hereditary autosomal dominant II. Acquired A. DIC B. Liver disease C. During post-operative period D. Anticoagulant therapy Slide 56 56 Clinical Manifestations Superficial thrombophlebitis Venous thromboses in adolescents or young adults Arterial thromboses rarely observed Skin necrosis during onset of oral anticoagulant therapy Slide 57 57 Protein S Cofactor for Protein C Vitamin K-dependent protein Enhances binding of Protein C to phospholipid surfaces Slide 58 58 Fibrinolysis/ Inhibitor Assays Plasminogen Activity Chromogenic Substrate Antithrombin III Antigen RID, Elisa Activity Chromogenic Substrate Protein C Antigen EID, Elisa Activity Chromogenic Substrate Functionality Clot-based Assay Slide 59 59 Acquired Inhibitors VIIILupus InhibitorAnticoagulant APTTIncreasedIncreased PTNormalIncreased or Normal APTT:Increased,Increased, 50/50 mix withtime dependentnot time dependent normal plasma OtherVarying results with Characteristics different dilutions Factor XII, XI, IX and VIII Assays Slide 60 60 Hemostasis Homeostasis Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins BleedingClotting Slide 61 61 Routine Screening Tests Coagulation System PT Extrinsic System APTT Intrinsic System Fibrinogen Platelet Studies Count Aggregation Bleeding Time Fibrinolysis / Inhibitors Thrombin Clotting Time FDP Specific Assays AT-III, Protein C, Protein S Slide 62 62