Principles of Hemostasis

Transcription

1 Principles of Hemostasis Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH Revised: mmvi 1

2 Topics for Discussion Introduction Virchow s Triad Primary Hemostasis Secondary Hemostasis Physiologic Coagulation Laboratory Coagulation Fibrinolysis 2

3 Virchow s Triad 3

4 Virchow s Triad Changes in blood coagulability Platelets, Coagulation Factors & Inhibitors, Fibrinolysis Changes in vessel wall Endothelial changes due to inflammation or atherogenesis Changes in blood flow Rheology in vessels 4

5 Vessel Wall Tunica intima Endothelium - inner most layer of cells that separate the remainder of the vessel from the lumen Basement membrane thin layer of spongy connective tissue that secretes elastic collagen Tunica adventitia Tunica intermedia Surrounds the tunica intima Smooth muscle layer of smooth muscle cells that are under involuntary control and can dilate or constrict Connective tissue produces collagen fibers whose elasticity is reduced by hypertension Surrounds the tunica media Connective tissues produce elastic and non-elastic collagen fibers Prevents ballooning of vessel with high systolic blood pressure Aneurysm weaknesses in the tunica adventitia 5

6 Rheology Region 1 In this range of shear rates: Cells are in large aggregates (rouleaux formation) As shear rate increases, size of aggregates diminish Viscoelasticity is strongly influenced by aggregation tendency of RBCs Adapted from : /tech10.html Region 2 As shear rate increases: Cells are disaggregating Applied forces (yield stress) are forcing cells to orient and deform Blood viscosity decreases Region 3 With increasing stress: Cells deform With normal deformability, cells will form layers that slide on layers of plasma and align in the direction of flow Science of the deformation and flow of matter Blood is a fluidized suspension of elastic cells that demonstrates both a viscous and elastic effect Elastic effect makes blood a non-newtonian fluid (plasma is a Newtonian fluid) Blood has a yield stress that depends on hematocrit and fibrinogen concentration 6

7 Shear & Vessel Wall Endothelial Cells At Wall Surface: Greatest Shear Rate & Least Velocity Platelet Flowing Blood At Vessel Center: Greatest Velocity & Least Shear Rate Shear Stress Pressure- induced Force Between 2 Laminae Platelet Laminae (Arrow Length = Velocity) Shear Flow Endothelial Cells 7

8 Hemostasis Process by which blood is maintained in a fluid state and confined to the circulatory system Goal is to stop bleeding and to do so only at the site of injury Components Platelets Involved in Primary Hemostasis Coagulation system Involved in Secondary Hemostasis Fibrinolytic system Inflammatory processes Wound healing processes Platelet / fibrin mesh 8

9 Primary Hemostasis 9

10 Primary Hemostasis First physiological response to vascular injury, which is mediated by platelets, in order to arrest bleeding Mechanism Activation of platelets via stimulators such as thrombin Adhesion of platelets to subendothelium via interaction between GPIb and von Willebrand Factor (VWF) Release of platelet granule products in order to recruit more platelets to the injured site Aggregation of platelets via interaction between GPIIb/IIIa (α IIb β 3 ) and fibrinogen to form the initial plug Triggers secondary hemostasis (coagulation proteins) Affected by medications, platelet function status, and vessel wall status 10

11 Platelets Disk-shaped cell fragments produced in the bone marrow by megakaryocytes Life span ~ 10 days Glycoprotein Ib (GPIb) is involved in adhesion Glycoprotein IIb/IIIa (GPIIb/IIIa) is the primary receptor for fibrinogen (aggregation phase) Provide procoagulant surface on which coagulation proteins can interact Alpha granules Coagulation proteins GPIIb/IIIa Dense granules GPIb 11

12 Platelet Adhesion Platelet GPIb Endothelium VWF Injury site collagen exposed COL COL COL COL COL VWF VWF VWF Subendothelial extracellular matrix VWF 12

13 Platelet Aggregation Activated Platelet (Granule contents released) GPIb VWF GPIIb/IIIa Fibrinogen COL COL COL COL COL VWF VWF VWF Subendothelial extracellular matrix VWF 13

14 Tests for Primary Hemostasis Bleeding Time Assesses all components of Virchow s triad in vivo test performed directly on patient Has fallen into disrepute and replaced by instruments that perform in vitro bleeding times Platelet Aggregation studies Measure ability of platelets to aggregate, in vitro, when subjected to various stimulators (agonists) Predominantly assesses function of platelet glycoprotein IIb/IIIa receptor Von Willebrand Factor (VWF) assays Measure amount and function of VWF, a protein that works with platelets so that they adhere to site of injury Assesses function of VWF ligand in its interaction with platelet glycoprotein Ib receptor 14

15 Platelet Appearance / Function Electron Micrographs Aggregation Tracings Resting platelets Normal platelet function Aspirin-like defect Activated platelets 15

16 Secondary Hemostasis 16

17 Secondary Hemostasis Process of blood coagulation Mechanism Coagulation proteins work in concert to generate thrombin Thrombin converts fibrinogen to fibrin Fibrin consolidates the platelet plug made in primary hemostasis such that a thrombus (secondary hemostatic plug) is formed Prevents further blood loss from the injury site Credit: Weisel JW. University of Pennsylvannia 17

18 Coagulation Factors Factor XII (FXII) Factor XI (FXI) Factor X (FX) Factor IX (FIX) Factor VIII (FVIII) Factor VII (FVII) Factor V (FV) activated FXII (FXIIa) activated FXI (FXIa) activated FX (FXa) activated FIX (FIXa) activated FVIII (FVIIIa) activated FVII (FVIIa) activated FV (FVa) Factor II (prothrombin) is converted to thrombin (FIIa) Factor I (fibrinogen) is converted to fibrin 18

19 in vivo v in vitro Coagulation Physiologic ( in vivo ) coagulation is dependent upon the tissue factor pathway Goal is to form a thrombus Laboratory ( in vitro ) coagulation is dependent upon the contact system Classical waterfall or cascade concept Step-by-step biochemical reactions in which an inactive proenzyme is converted to a reactive enzyme which, in turn, converts another proenzyme to its active form Amplification process (very minute amounts of Factor XII yield large amounts of thrombin) Goal is to form a clot 19

20 Waterfall Scheme of Coagulation FXII FXIIa FXI FXIa FIX FVIIIa Ca ++ PL FIXa FVIIa FVII FX Ca ++ PL FXa TF FX FII Ca ++ FVa PL Thrombin TF = Tissue Factor After Macfarlane RG. Nature 1964;202:498-9 Fibrinogen Fibrin Ca + + = Calcium ion PL = Phospholipid 20

21 Physiologic Coagulation Thrombus Formation 21

22 Monroe DM, et al. ATVB 2006;26:41-8 Initiation Phase Extrinsic Pathway 22

23 Monroe DM, et al. ATVB 2006;26:41-8 Amplification Phase 23

24 Monroe DM, et al. ATVB 2006;26:41-8 Propagation Phase Intrinsic Pathway 24

25 Recapping Secondary Hemostasis Tissue Factor complexed with FVIIa initiates coagulation at site of injury Small amounts of thrombin are generated that activate platelets Coagulation factors form complexes on platelet surfaces Very large amounts of thrombin are formed to convert fibrinogen to fibrin Fibrin reinforces platelet plug (primary hemostasis) and hemostasis is achieved 25

26 Laboratory Coagulation Clot Formation 26

27 First the Specimen Blood is collected into a tube that contains sodium citrate, an anticoagulant Blood fluidity is maintained because sodium citrate binds calcium ions, which are critical to the coagulation process Tube is centrifuged in order to separate plasma from buffy coat (white blood cells & platelets) and red blood cells Plasma is used for testing PLASMA contains FIBRINOGEN Serum does not contain Fibrinogen Plasma is platelet poor since platelets remain in buffy coat Plasma Buffy Coat Red Cells 27

28 Coagulation in the Laboratory Intrinsic Pathway Extrinsic Pathway XII XI IX VII Tissue Factor APTT Intrinsic + Common VIII Common X V II Pathway PT Extrinsic + Common Fibrinogen Fibrin Clot 28

29 Routine Coagulation Assays Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT) Quantitative Fibrinogen (FIB) Thrombin Time (TT) Assays for specific coagulation factors Factors assessed by a PT-based test system: FVII, FV, FX, and FII Factors assessed by an APTT-based test system: FXII, FXI, FIX, and FVIII 29

30 Prothrombin Time (PT) Time for clot formation ~ 12 seconds Incubate at 37 o C for ~3 minutes 0.1 ml Thromboplastin + Ca ml Plasma PT Reagent Composition Thromboplastin Tissue Factor (recombinant/human or animal brain) Credit: PNAS; Collet JP and Weisel JW. Un Pennsylvannia Lipid (source of phospholipid since platelets were removed from plasma) CaCl 2 used to reintroduce calcium ions that were chelated by sodium citrate Historically referred to as complete since both phospholipid and apoprotein make up the reagent 30

31 Causes for Prolonged PT Deficiencies or abnormalities in: FVII (Extrinsic Pathway) FV, FX, FII (prothrombin), and FI (fibrinogen) Both PT and APTT will be prolonged Vitamin K antagonists PT sensitive to reductions in three of four vitamin K-dependent procoagulant proteins: FVII, FX, and FII FIX measured by APTT Pharmacologic anticoagulants that modify vitamin K-dependent proteins such that they do not bind calcium thereby reducing blood coagulability Liver disease Site for synthesis of vitamin K-dependent proteins Site for clearance of coumarins (warfarin) and coagulation proteins 31

32 Biochemistry: Vit K-Dep Proteins Addition of an extra carboxyl group to glutamate (Glu) residues at their amine termini gives rise to a novel amino acid called gamma-carboxyglutamate (Gla) Presence of Gla enables proteins to undergo a calciumdependent conformational change that allows for their binding to phospholipid surfaces and generation of membrane bound macromolecular complexes 32

33 Vitamin K Antagonists (AVK) DICUMAROL: 3,3 -methylenebis (4-hydroxycoumarin) Isolated by Karl Link (University of Wisconsin-1939) as the antivitamin K agent responsible for hemorrhagic disorder in cattle Coumarin derivatives Bishydroxycoumarin (Dicumarol) Warfarin (Coumadin ) Analog #42 of many coumarins synthesized by Dr Link and named by him as WARFarin for the Wisconsin Alumni Research Foundation and coumarin Require monitoring because: Vitamin K-dependent proteins have different half-lives Differences in drug absorption and clearance Levels affected by concomitant medications, comorbid conditions, changes in diet, patient compliance PT (thromboplastin) reagents vary in their reaction to clotting defects produced by AVK (warfarin) 33

34 Warfarin Sodium Oral anticoagulant of choice in North America Pharmacologic properties more favorable than Dicumarol Warfarin is 5-10x more potent Racemic mixture (~1:1) of R & S isomers S isomer is 5x more potent than R isomer Hepatic microsomal enzyme cytochrome P450 2C9 is responsible for the oxidative metabolism of S isomer Bioavailability Absorbed from gastrointestinal tract Maximal blood concentrations reached in 90 minutes Half-life (T½) is hours At therapeutic concentrations, 99% of warfarin is bound to albumin and 1% is free and can bind to its receptor on hepatic cells Anticoagulant effects are reversed by administration of vitamin K or biologic products that contain vitamin K-dependent proteins 34

35 Warfarin Mode of Action Des-Carboxy Prothrombin ( Glu ) CO 2 O 2 γ-carboxylase H 2 O Prothrombin ( Gla ) Vitamin K hydroquinone (active form K 1 H 2 ) Vitamin K epoxide reductase (VKOR) Vitamin K 2, 3 epoxide (inactive form K 1 O) Regeneration of active form is sensitive to warfarin NAD + WARFARIN NADH 35

36 International Normalized Ratio INR = Patient PT Mean Normal PT ISI Normalizes the PT by mathematically considering differences in PT reagents (thromboplastins) Only to be used to monitor long term anticoagulant effects for patients stabilized on oral anticoagulant therapy Prevent recurrence of thrombosis caused by under anticoagulation Prevent hemorrhagic complications caused by over anticoagulation 36

37 Activated Partial Thromboplastin Time Time for clot formation ~ 30 seconds 0.1 ml CaCl 2 Incubate at 37 o C for ~5 minutes 0.1 ml Activator 0.1 ml Plasma APTT Reagent Composition Activator to convert FXII to FXIIa Phospholipid (replaces in vivo platelet surface on which coagulation reactions occur) CaCl 2 used to reintroduce calcium ions that were chelated by sodium citrate Referred to as partial thromboplastin since no Tissue Factor is used Two-stage assay (activation and re-calcification) 37

38 Intended Use for APTT Screening test Intrinsic (and severe common pathway) factor deficiencies Laboratory monitoring Unfractionated heparin Other antithrombotic agents (Direct Thrombin Inhibitors) Laboratory detection of the Lupus Anticoagulant 38

39 Causes for Prolonged APTT Most common causes Heparin (contamination from lines or therapeutic) Lupus Anticoagulant Other causes Deficiencies of coagulation factors FVIII (Hemophilia A or Von Willebrand Disease), FIX, FXI, FXII Liver disease (site of production for most coagulation factors) Consumption of coagulation factors as seen in Disseminated Intravascular Coagulation (DIC) 39

40 Heparin Heparin is a heterogeneous group of straight-chain anionic mucopolysaccharides (glycosaminoglycans) Molecular weights range from 5 40 kilodaltons Composed of alternating D-glucosamine residues linked 1 4 to either L-iduronic acid or D-glucuronic acid Heparin is highly acidic therefore binds to positively charged amino acids such as arginine & lysine Pentasaccharide sequence Comprisies ~30% of heparin Binds to antithrombin (AT) Accelerates AT inhibition of activated factors XII, XI, IX, X, and II (thrombin) thus serving as an anticoagulant Pharmaceutical heparins are extracted from pig intestinal mucosa (source of mast cells) 40

41 Heparin and Antithrombin 41

42 APTT Monitoring of Heparin Assumes antithrombotic (anti-iia) effect parallels anticoagulant effect Limitations Pre-treatment APTT of patient Baseline APTT of patient prolonged due to Lupus Anticoagulant Baseline APTT of patient sample below or at low end of reference interval due to high levels of FVIII (apparent heparin resistance ) APTT reagents vary in sensitivity to heparin Laboratories must determine responsiveness of their APTT reagent to unfractionated heparin Determine APTT therapeutic interval (seconds) for reagent used to monitor heparin therapy 42

43 Direct Thrombin Inhibitors Hirudin [Lepirudin (rdna) trade name: Refludan ] Approved in USA, Canada, and EU for Heparin Induced Thrombocytopenia (HIT) complicated by thrombosis Target APTT is x patient baseline APTT In absence of severe thrombosis, some experts recommend a target APTT of x patient baseline APTT and monitor every 4 hours Argatroban [non-us trade name: Novastan] Approved for HIT with or without thrombosis and also for anticoagulation during percutaneous coronary intervention (PCI) in patients with, or at risk for, HIT Target APTT x patient baseline APTT (maximum 100 seconds) Hirulog [Bivalirudin-trade name: Angiomax ] Undergoing evaluation for use as an anticoagulant for on-pump and off-pump cardiac surgery in patients with HIT Target APTT is x patient baseline APTT 43

44 Recap ping the PT and APTT PT and APTT are screening assays to determine if a patient, when challenged, has a potential to bleed If warfarin or heparin are not present in sample, then: Prolonged PT and normal APTT = deficiency of FVII Normal PT and prolonged APTT = deficiencies in any of the intrinsic pathway factors (FVIII, FIX, FXI, or FXII) Prolongation of both PT and APTT = deficiencies of factors common to both pathways (FX, FV, FII, or fibrinogen) PT, via the INR, is used to monitor oral anticoagulant therapy (warfarin) APTT is used to monitor heparin anticoagulant therapy APTT is affected by inhibitors such as Lupus Anticoagulant 44

45 Tests for Fibrinogen Quantitative Fibrinogen Measures the amount of fibrinogen present in plasma Low levels, termed hypofibrinogenemia, can be inherited but generally are due to acquired causes such as DIC, liver disease, or fibrinolytic therapy High levels are seen in inflammatory states since fibrinogen is an acute phase reactant Thrombin Time (TT) Assesses the functionality of fibrinogen in plasma TT clotting time prolonged HEPARIN Direct thrombin inhibitors Hypofibrinogenemia Dysfibrinogenemia Elevated fibrin split products Time for clot formation ~ 15 seconds Incubate at 37 o C for ~2 minutes 0.2 ml Diluted Thrombin 0.2 ml Plasma 45

46 Quantitative Fibrinogen Time for clot formation ~ 5-14 seconds Incubate at 37 o C for ~3 minutes 0.1 ml Thrombin Calibrator plasma is serially diluted (1:5, 1:10, 1:20, and 1:40) to establish a reference curve (see graph below) Patient plasma is diluted 10 fold (1:10) in buffer 0.2 ml Diluted Plasma Patient clotting time in seconds is read against the reference curve Patient clotting time of 8.0 seconds equates to 281 mg/dl fibrinogen in example at right Fibrinogen concentration is inversely proportional to clotting time Clotting Time-Seconds Patient mg/dl Fibrinogen 46

47 Coagulation: A Balancing Act Generate Thrombin Inhibit Thrombin Formation 47

48 Inhibitors Naturally occurring inhibitors Protein C (activated) and Protein S Inhibit coagulation cofactors FVIIIa and FVa Antithrombin Inhibits FXIa, FIXa, FXa, FVIIa/TF, and thrombin (IIa) Pathologic inhibitors Acquired or autoimmune antibodies to specific coagulation factors Pharmacologic inhibitors Heparin and Low Molecular Weight Heparin Warfarin Direct Thrombin Inhibitors 48

49 Fibrinolysis 49

50 Hemostasis: A Delicate Balance Generates Thrombin Generates Plasmin Form a Thrombus Dissolve a Thrombus 50

51 Fibrinolytic System FXIII Fibrinogen Fibrin(ogen) Degradation Products Thrombin FIBRIN TM TAFIa Plasmin TAFI tpa Antiplasmin PC Thrombin TM APC Plasminogen PAI

52 Breakdown of Fibrin(ogen) Fibrinogen FPB FPA & B Fibrinogen Fragment X Thrombin Fibrin Monomer Fibrin Clot DD YY FXIII Fibrin Polymer FXIIIa DD EE DD PLASMIN DD EE DD DD EE DD D-dimer DD DD EE DD DD EE DD 52

53 Fibrinolytic Agents All currently available thrombolytic agents are plasminogen activators (PA) Convert patient plasminogen to plasmin which then acts on fibrin within a thrombus Additionally can breakdown fibrinogen (fibrinogenolysis) Commonly referred to as the lytic state (systemic lysis) Therapeutic doses of PA overwhelm PAI-1 and α 2 -antiplasmin Beneficial effect is reduction of thrombus size (thrombolysis) Negative effect is that hemostatic plugs are also lysed Most commonly used agents are: Streptokinase (SK), Alteplase (tpa), Reteplase, and Tenecteplase (TNK-tPA) 53

54 Summaries 54

55 Time Frame for Hemostasis Platelets Primary Hemostasis Vessel constriction occurs immediately Platelet adhesion occurs in in seconds Platelet aggregation takes minutes Coagulation Factors Secondary Hemostasis Activation of of coagulation factors occurs in in seconds Fibrin forms in in minutes Fibrinolytic Proteins Fibrinolysis Activation of of fibrinolytic proteins happens immediately Dissolving the the thrombus requires hours 55

56 Bleeding: Balance is Disrupted Presence of Inhibitors* Pharmacologic (warfarin, heparin) Allo or Auto-antibodies to Factors 56

57 Thrombosis: Balance is Disrupted Inhibitors* Activated Protein C Protein S Antithrombin 57

58 Conclusion Primary hemostasis, a platelet-dependent process, forms hemostatic plugs when a vessel is injured Secondary hemostasis, a coagulation factor-dependent process, begins with Tissue Factor exposure Small amounts of thrombin are generated via FXa formation by the TF:FVIIa complex ( Extrinsic Pathway ) Sustained thrombin generation depends on FXa formation via FIXa and FVIIIa-mediated complexes on an activated platelet surface Amount of thrombin generated dictates bleeding or thrombotic risk The clinical history is the best test for hemostasis 58

59 References Ansell J, et al. The pharmacology and management of the vitamin K antagonists: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:204S-233S. Clinical and Laboratory Standards Institute (CLSI). One-stage Prothrombin Time (PT) test and Activated Partial Thromboplastin Time (APTT) test; Approved Guideline H47-A, Crowther MA, et al. Practical aspects of anticoagulant therapy (Chapter 89). In: Colman RW, ed. Hemostasis and Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, Fairweather RB, et al. College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy: Laboratory monitoring of oral anticoagulant therapy. Arch Pathol Lab Med 1998;122(9): Greaves M, Preston FE. Approach to the bleeding patient (Chapter 48). In: Colman RW, ed. Hemostasis and Thrombosis, 4 th ed. Philadelphia: Lippincott Williams & Wilkins, Hirsh J, Raschke R. Heparin and low-molecular-weight heparin: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:188S-203S. Konkle BA. Clinical approach to the bleeding patient (Chapter 77). In: Colman RW, ed. Hemostasis and Thrombosis, 5 th ed. Philadelphia: Lippincott Williams & Wilkins, Link KP. The discovery of Dicumarol and its sequels. Circulation 1959;19(1): Olson JD, et al. College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy: Laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med 1998;122(9): Physicians Desk Reference, 60th ed. Montvale: Thomson PDR, Poller L. Prothrombin Time (Chapter 6) and Activated Partial Thromboplastin Time (Chapter 5). In: Jespersen J, ed. Laboratory Techniques in Thrombosis-A Manual, 2nd ed. Dordrecht: Kluwer Academic Publishers, Tran HAM, Ginsberg JS. Anticoagulant therapy for major arterial and venous thromboembolism (Chapter 116). In: Colman RW, ed. Hemostasis and Thrombosis, 5th ed. Philadelphia: Lippincott Williams & Wilkins, van den Besselaar, AMHP, Gralnick HR, Lewis SM, Editors. Thromboplastin Calibration and Oral Anticoagulant Control. Dordrecht: Martinus Nijhoff Publishers,