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Functional Aspects of.

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Presentation on theme: "Functional Aspects of."— Presentation transcript:

1 Functional Aspects of

2 Homeostasis: Vessels Coagulation Proteins Platelets
State of fluid equilibrium within the blood vessels Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins

3 Fibrinolysis/Inhibitors
Hemostasis Homeostasis Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins Bleeding Clotting

4 Platelets Fibrinolysis/Inhibitors Coagulation Proteins Vessels

5 Vascular System Endothelial Cells Basement Membrane Red Blood Cells
Platelets White Cells

6 Platelets Fibrinolysis/Inhibitors Coagulation Proteins Vessels

7 What are Platelets? Disk-shaped “cells”
produced in the megakaryocytes of the bone marrow Mature Platelet Megakaryocyte Bone Marrow

8 Anatomy of a Platelet Dense Body ADP Serotonin ATP Calcium
Microtubular System Glycogen Exterior Coat (Glycocalyx) Mitochondria Surface Connecting System Cytosol Factor XIII Alpha Granules PF4 Fibrinogen Factor V Fibronectin Factor VIII R:ag b Thromboglobulin (vWF) Plasma Membrane PF3 Dense Tubular System

9 Storage and Circulation
Quantity - 200, ,000/mm3 Life Span - 10 days 33% pooling 67% in the circulation Megakaryocyte Spleen

10 Platelet Function Adhesion Aggregation Coagulation ADP Release A
Shape Change Release Fibrin Formation A B C 3 seconds 10 seconds 5 minutes ADP Release Adhesion

11

12

13 Platelet Testing Peripheral Smear Platelet Count Platelet Aggregation
Bleeding Time

14 Platelet Aggregation + Platelet Rich Plasma (PRP) Aggregating Reagent
Aggregate Clumping Baseline Light Transmission Increased Light Transmission +

15 Typical Biphasic Pattern
100 90 80 70 60 50 40 30 20 10 Platelet Rich Plasma Poor Secondary Response (Release) Primary Response Injection Point Lag

16 Bleeding Time Cut 1 mm deep 5 mm long Constant Pressure
Expected Range minutes 40 mm

17 Platelets Coagulation Proteins Fibrinolysis/Inhibitors Vessels

18 Coagulation Factors Factor I Fibrinogen Factor II Prothrombin
Factor III Tissue Thromboplastin Factor IV Calcium Ions Factor V Labile Factor, Proaccelerin Factor VII Stable Factor, Proconvertin Factor VIII Antihemophilic Factor Factor IX Christmas Factor Factor X Stuart-Prower Factor Factor XI Plasma Thromboplastin Antecedent Factor XII Hageman Factor Factor XIII Fibrin Stabilizing Factor

19 VII X IX XI XII XIIa XIa IXa VIIIa Xa VIIa VIII II Va V IIa XIII XIIIa
Fibrinogen VII Extrinsic Pathway (PT) Tissue Factor Kinins X IX XI XII HMWK Prekalli- krein Kalli- XIIa XIa IXa VIIIa Xa VIIa Pro- Urokinase Intrinsic (aPTT) Fibrinolysis VIII Ure- Kinase T-PA Plas- minogen Plasmin Common II Va V IIa XIII XIIIa Fibrin Polymer Fibrin Clot + Platelet Plug

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)

21 Fibrinogen Degradation Fibrin Stabilizing Factor (FSF)
Biosynthesis: Liver MW: 340,000 daltons Plasma Concentration: 2500 mg/L In Vivo Half-Life: 20 hours Pathology: Afibrinogenemia, autosomal recessive. Dysfibrinogenemia, autosomal dominant Fibrinogen Factor I Factor IIa Fibrinopeptide A Fibrinopeptide B Fibrinogen Factor II Fibrin Monomer Procoagulant Activation Polymer CA+ XIII XIIIa Soluble Plasmin Anticoagulant Inhibition (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

22 Factor V Proaccelerin, Labile Factor Factor IIa V Factor Va Activated
Biosynthesis: Liver, megakaryocytes MW: 330,000 daltons Plasma Concentration: 5-12 mg/L In Vivo Half-Life: 25 hours Pathology: Parahemophilia, autosomal recessive Factor V Proaccelerin, Labile Factor Factor IIa V Activated Protein C Protein S Phospholipid (Platelet Factor 3) Inactive Fragments Ca++ Factor Va Complex Activation Procoagulant Anticoagulant Inhibition

23 Factor VIII vWF Antihemophilic Factor von Willebrand Factor Factor IIa
Biosynthesis: Liver, endothelium; Factor VIII Related Antigen, megakaryocyte MW(FVIII + vWF): 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 Willebrand’s disease, autosomal dominant Factor IIa Factor Xa or Procoagulant Factor VIII Activation Factor VIIIa Inhibition Inactive Fragments Anticoagulant Protein C Complex Activated Protein C Protein S Phospholipid (Platelet Factor 3) Ca++

24 *or Heparin Cofactor 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 IIa Anti- thrombin III Heparin * Activation Fragments

25 Factor VII Proconvertin, Stable Factor High Ca++ Molecular Weight
Biosynthesis: Liver, Vitamin K dependent MW: 55,000 daltons Plasma Concentration: 1 mg/L In Vivo Half-Life: 5 hours Pathology: Hypoproconvertinemia, autosomal recessive Ca++ Phospholipid (Platelet Factor 3) High Molecular Weight Kininogen Factor VIIa Tissue Xa TFPI* XIIa IXa or Procoagulant Activation Anticoagulant Inhibition VII *Tissue Factor Pathway Inhibitor (a.k.a. LACI or EPI)

26 Factor IX Christmas Factor Ca++ Tissue Factor VIIa XIa or Procoagulant
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 IXa Factor IX Activation Procoagulant Anticoagulant Inhibition XIa or Anti- thrombin III Heparin

27 Phospholipid (Platelet
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 Factor VIIIa Tissue Phospholipid (Platelet Factor 3) IXa VIIa Factor X Factor Xa Anti- thrombin III Heparin Ca++ Activation Procoagulant Anticoagulant Inhibition or Extrinsic X-ase Complex Intrinsic Tissue Factor Factor III, Thromboplastin Biosynthesis: Brain, lung, subendothelium MW: 45,000 daltons

28 Factor XI Plasma Thromboplastin Antecedent Ca++ High Factor Molecular
Biosynthesis: Liver MW: 158,000 daltons Plasma Concentration: 4 mg/L In Vivo Half-Life: 65 hours Pathology: Hemophilia C, autosomal recessive Factor XI Plasma Thromboplastin Antecedent Ca++ High Molecular Weight Kininogen Factor XIIa XIa Factor XI Activation Procoagulant Anticoagulant Inhibition a1- Anti- trypsin

29 Prekallikrein Fletcher Factor Ca++ High Factor Molecular XIIa Weight
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- Activation Procoagulant Anticoagulant Inhibition C1 Esterase Inhibitor

30 Factor XII Hageman Factor Negatively Charged Activating Surface
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 Negatively Charged Activating Surface (e.g., kaolin, ellagic acid silica) Kalli- krein Factor XII Kinins High Blood Pressure and Inflammation XIIa HMWK C1 Esterase Inhibitor * FXIIf alter vascular permeability Factor XII Fragments* Activation Procoagulant Anticoagulant Inhibition High Molecular Weight Kininogen (HMWK) Fitzgerald Factor MW: 120,000 daltons Plasma Concentration: 70 mg/L Pathology: Fitzgerald trait, autosomal recessive

31 VII VIIa X Xa II Va IIa V XIII XIIIa Extrinsic Pathway (PT) Common
Fibrinigen VII X Xa VIIa II Va V IIa XIII XIIIa Fibrin Polymer Tissue Factor Fibrin Clot + Platelet Plug

32 Prothrombin Time Factors I II V VII X Thromboplastin and Calcium
Patient’s Plasma Factors I II V VII X

33 Intrinsic Pathway (aPTT) Common
Fibrinogen Kinins X IX XI XII HMWK Prekalli- krein Kalli- XIIa XIa IXa VIIIa Xa Intrinsic Pathway (aPTT) VIII Common II Va V IIa XIII XIIIa Fibrin Polymer Fibrin Clot + Platelet Plug

34 Activated Partial Thromboplastin Time
Ca++ Patient’s Plasma Factors I II V VIII IX X XI XII Phospholipid and Activator

35 Quantitative Fibrinogen
High concentration of thrombin 1:10 dilution patient’s plasma Time (in seconds) Fibrinogen in mg/dL 20 40 60 100 200 400 600 30 10 6 3 1:40 1:30 1:20 1:10 1:5

36 Thrombin Clotting Time
Low concentration of thrombin Undiluted patient’s plasma Screens for effects of • Heparin • FDPs

37 Monitoring Anticoagulant Therapy
Vitamin K Antagonists Antithrombin III Accelerators

38 Vitamin K Antagonists • Coumarin, Dicumarol, Warfarin
• Oral Administration • Long-Acting Effect • Factors IIa, VIIa, IXa, Xa • PT

39 AT-III Accelerators • Heparin, Heparinoids, LMW Heparin
• Parenteral Administration • Short-Acting Effect • Factors IIa, IXa, Xa • APTT

40 Heparin/AT-III Complex

41 Variables in Monitoring Heparin
• Time of Specimen Collection • Type of Anticoagulant • Centrifugation of Specimen • Instrumentation and Reagents

42 Factor Assays Diagnosis Therapy

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 Standard or patient’s plasma dilution 0.2 mL Thromboplastin Calcium Chloride Mixture

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 patient’s plasma dilution 0.1 mL Phospholipid and Activator 0.1 mL Calcium Chloride

45 The Standard Curve Dilution % Activity Clotting Time
1:5 20.0% 14.3 seconds 1: % 17.3 seconds 1:20 5.0% 21.0 seconds 1:40 2.5% 26.0 seconds 30 25 20 15 10 5

46 Minimum Plasma Levels Minor Major Factor Spontaneous Trauma Hemorrhage or Surgery I Fibrinogen mg/dL II % V % VII % VIII % Hemophilia A % von Willebrand 25 25% IX % X % XI % XII 10 10% XIII 1 5% Williams, W. J., Hematology, 1972

47 Fibrinolysis/Inhibitors
Vessels Coagulation Proteins Platelets Fibrinolysis/Inhibitors

48 Fibrinolysis Activators Plasminogen Plasmin Fibrinogen Fibrin
Degradation Products R.E.S.

49 Plasminogen tPA or or Kallikrein Urokinase Plasminogen Plasmin Plasmin
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 Plasminogen tPA (tissue Plasminogen Activator) or or Kallikrein Urokinase Anticoagulant Plasminogen Activation Plasmin Procoagulant Inhibition Plasmin a2 - Anti- plasmin a2 - Anti- plasmin

50 Thrombolytic Therapy • Anaphylaxis • GI Bleeding
• t-PA • Streptokinase • Urokinase • Anaphylaxis • GI Bleeding • Intracranial Hemorrhage

51 Heparin Thrombin Complex
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 (Inhibited) Heparin Thrombin Complex (Complexed)

52 Antithrombin-III Decreased Levels 1. Congenital
2. Acquired – decreased synthesis 3. Acquired – increased utilization 4. Drug-induced

53 Protein C • Vitamin K-dependent plasma protein
• Inactivates Factors V and VIII • Stimulates fibrinolysis

54 Protein C Thrombo- modulin Factor IIa Protein C Activation Peptide
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 (?) Protein C Thrombo- modulin Factor IIa Protein C Activation Peptide Protein C Anticoagulant Activation Activated Protein C* Inhibition Activated Protein C Procoagulant * Requires Protein S for functional activity Protein C Inhibitor Protein C Inhibitor

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

56 Clinical Manifestations
• Superficial thrombophlebitis • Venous thromboses in adolescents or young adults • Arterial thromboses rarely observed • Skin necrosis during onset of oral anticoagulant therapy

57 Protein S • Cofactor for Protein C • Vitamin K-dependent protein
• Enhances binding of Protein C to phospholipid surfaces

58 Fibrinolysis/ Inhibitor Assays
Plasminogen Activity – Chromogenic Substrate Antithrombin III Antigen – RID, Elisa Protein C Antigen – EID, Elisa Functionality – Clot-based Assay

59 Acquired Inhibitors VIII Lupus Inhibitor Anticoagulant
APTT Increased Increased PT Normal Increased or Normal APTT: Increased, Increased, 50/50 mix with time dependent not time dependent normal plasma Other Varying results with Characteristics different dilutions Factor XII, XI, IX and VIII Assays

60 Fibrinolysis/Inhibitors
Hemostasis Homeostasis Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins Bleeding Clotting

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

62


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