Hemostasis principles


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Hemostasis principles

  1. 1. Principles of Hemostasis Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SHRevised: mmvi 1
  2. 2. Topics for Discussion I nt ro d Vi r uct cho ion P ri w’s ma Tri ry Sec on He ad Ph dar mos ysi yH tas Lab olog em is o ra ic C ost Fib tor oag asis rin yC ol y sis oag ulatio ul a n tio nwww.CLOT-ED.com 2
  3. 3. Virchow’s Triadwww.CLOT-ED.com 3
  4. 4. Virchow’s Triad Changes in blood coagulability Platelets, Coagulation Factors & Inhibitors, Fibrinolysis Changes in vessel wall Changes in blood flow Endothelial changes due to Rheology in vessels inflammation or atherogenesiswww.CLOT-ED.com 4
  5. 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 Surrounds the tunica media Connective tissues – produce elastichttp://www.alegent.com/122711.cfm and non-elastic collagen fibers Tunica intermedia Prevents ballooning of vessel with high systolic blood pressure Surrounds the tunica intima Aneurysm – weaknesses in the tunica Smooth muscle – layer of smooth muscle cells adventitia that are under involuntary control and can dilate or constrict Connective tissue – produces collagen fibers whose elasticity is reduced by hypertensionwww.CLOT-ED.com 5
  6. 6. RheologyRegion 1 Region 2In this range of shear rates: As shear rate increases: Cells are in large Cells are disaggregating aggregates (rouleaux Applied forces (yield formation) stress) are forcing cells to As shear rate increases, orient and deform size of aggregates diminish Blood viscosity decreases Viscoelasticity is strongly influenced by aggregation tendency of RBCs Region 3 With increasing stress: Cells deform With normal deformability, cells will form layers thatAdapted from : slide on layers of plasmahttp://www.vilastic.com and align in the direction/tech10.html 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 concentrationwww.CLOT-ED.com 6
  7. 7. Shear & Vessel Wall Endothelial Cells At Wall Surface: Greatest Platelet Flowing Blood Shear Rate & Least Velocity Shear At Vessel Center: Greatest Platelet Flow Velocity & Least Shear Rate Shear Stress Pressure- induced Force Between 2 Laminae Laminae (Arrow Length = Velocity) Endothelial Cellswww.CLOT-ED.com 7
  8. 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 Platelet / fibrin mesh – Inflammatory processes – Wound healing processeswww.CLOT-ED.com 8
  9. 9. Primary Hemostasiswww.CLOT-ED.com 9
  10. 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 (αIIbβ3) and fibrinogen to form the initial plug Triggers secondary hemostasis (coagulation proteins) Affected by medications, platelet function status, and vessel wall statuswww.CLOT-ED.com 10
  11. 11. Platelets Disk-shaped cell fragments produced in GPIIb/IIIa the bone marrow by megakaryocytes Alpha Dense Life span ~ 10 days granules granules 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 Coagulation can interact proteins GPIbwww.CLOT-ED.com 11
  12. 12. Platelet Adhesion Platelet GPIb Endothelium VWF Injury site collagen exposed COL COL COL CO L COL VWF VWF VWF VWFSubendothelial extracellular matrixwww.CLOT-ED.com 12
  13. 13. Platelet Aggregation Activated Platelet GPIb Fibrinogen (Granule contents released) VWF GPIIb/IIIa COL COL COL CO L COL VWF VWF VWF VWFSubendothelial extracellular matrixwww.CLOT-ED.com 13
  14. 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 receptorwww.CLOT-ED.com 14
  15. 15. Platelet Appearance / Function Electron Micrographs Aggregation Tracings Resting platelets Normal platelet Aspirin-like defect function Activated plateletswww.CLOT-ED.com 15
  16. 16. Secondary Hemostasiswww.CLOT-ED.com 16
  17. 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 Credit: Weisel JW. University of Pennsylvannia that a thrombus (secondary hemostatic plug) is formed Prevents further blood loss from the injury sitewww.CLOT-ED.com 17
  18. 18. Coagulation Factors Factor XII (FXII) activated FXII (FXIIa) Factor XI (FXI) activated FXI (FXIa) Factor X (FX) activated FX (FXa) Factor IX (FIX) activated FIX (FIXa) Factor VIII (FVIII) activated FVIII (FVIIIa) Factor VII (FVII) activated FVII (FVIIa) Factor V (FV) activated FV (FVa) Factor II (prothrombin) is converted to thrombin (FIIa) Factor I (fibrinogen) is converted to fibrinwww.CLOT-ED.com 18
  19. 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 clotwww.CLOT-ED.com 19
  20. 20. Waterfall Scheme of Coagulation FXII FXIIa FXI FXIa FVIIIa FIX Ca++ PL FIXa FVIIa FVII TF Ca++ PL FX FXa FX FVa FII Ca++ PL Thrombin TF = Tissue Factor Ca+ += Calcium ionAfter Macfarlane RG. Nature 1964;202:498-9 Fibrinogen Fibrin PL = Phospholipidwww.CLOT-ED.com 20
  21. 21. Physiologic Coagulation Thrombus Formationwww.CLOT-ED.com 21
  22. 22. Initiation Phase Monroe DM, et al. ATVB 2006;26:41-8 “Extrinsic Pathway”www.CLOT-ED.com 22
  23. 23. Amplification Phase Monroe DM, et al. ATVB 2006;26:41-8www.CLOT-ED.com 23
  24. 24. Propagation Phase Monroe DM, et al. ATVB 2006;26:41-8 “Intrinsic Pathway”www.CLOT-ED.com 24
  25. 25. Recapping Secondary HemostasisTissue Factor complexed with FVIIa initiates coagulation at site of injurySmall 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 achievedwww.CLOT-ED.com 25
  26. 26. Laboratory Coagulation Clot Formationwww.CLOT-ED.com 26
  27. 27. First the Specimen Blood is collected into Plasma is used for testing a tube that contains – PLASMA contains sodium citrate, an FIBRINOGEN anticoagulant – Serum does not contain Blood fluidity is Fibrinogen maintained because sodium citrate binds Plasma is “platelet poor” calcium ions, which are since platelets remain in critical to the buffy coat coagulation process Tube is centrifuged in order to separate plasma from buffy coat Plasma (white blood cells & Buffy Coat platelets) and red blood cells Red Cellswww.CLOT-ED.com 27
  28. 28. Coagulation in the Laboratory Intrinsic Pathway Extrinsic Pathway XII XI VII Tissue Factor IX APTT VIII PT Intrinsic X Extrinsic + Common V Pathway + Common II Common Fibrinogen Fibrin Clotwww.CLOT-ED.com 28
  29. 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 FVIIIwww.CLOT-ED.com 29
  30. 30. Prothrombin Time (PT) Time for clot formation ~ 12 seconds Incubate at 37 oC for ~3 minutes 0.1 ml Thromboplastin + Ca++ 0.1 ml PlasmaPT Reagent Composition Thromboplastin Credit: PNAS; Collet JP and − Tissue Factor (recombinant/human or animal brain) Weisel JW. Un Pennsylvannia − Lipid (source of phospholipid since platelets were removed from plasma) − CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate Historically referred to as “complete” since both phospholipid and apoprotein make up the reagentwww.CLOT-ED.com 30
  31. 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 proteinswww.CLOT-ED.com 31
  32. 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 calcium- dependent conformational change that allows for their binding to phospholipid surfaces and generation of membrane bound macromolecular complexeswww.CLOT-ED.com 32
  33. 33. Vitamin K Antagonists (AVK) DICUMAROL: 3,3’-methylenebis (4-hydroxycoumarin) – Isolated by Karl Link (University of Wisconsin-1939) as the anti- vitamin 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)www.CLOT-ED.com 33
  34. 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 36-42 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 proteinswww.CLOT-ED.com 34
  35. 35. Warfarin Mode of Action CO2 H2O O2 Des-Carboxy Prothrombin Prothrombin (“Glu”) γ-Carboxylase (“Gla”) Vitamin K Vitamin K hydroquinone Vitamin K 2, 3 epoxide(active form K1H2) (inactive form K1O) epoxide reductase (VKOR) Regeneration of active form NAD+ NADH is sensitive to warfarin WARFARINwww.CLOT-ED.com 35
  36. 36. International Normalized Ratio ISI Patient PT INR = Mean Normal PT “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 anticoagulationwww.CLOT-ED.com 36
  37. 37. Activated Partial Thromboplastin Time Time for clot formation ~ 30 seconds 0.1 ml CaCl2 Incubate at 37 oC for ~5 minutes 0.1 ml Activator 0.1 ml PlasmaAPTT Reagent Composition Activator to convert FXII to FXIIa Phospholipid (replaces “in vivo” platelet surface on which coagulation reactions occur) CaCl2 – 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)www.CLOT-ED.com 37
  38. 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 Anticoagulantwww.CLOT-ED.com 38
  39. 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)www.CLOT-ED.com 39
  40. 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)www.CLOT-ED.com 40
  41. 41. Heparin and Antithrombinwww.CLOT-ED.com 41
  42. 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 therapywww.CLOT-ED.com 42
  43. 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 1.5-2.5 x patient baseline APTT – In absence of severe thrombosis, some experts recommend a target APTT of 1.5-2.0 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 1.5-3.0 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 1.5-2.5 x patient baseline APTTwww.CLOT-ED.com 43
  44. 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 Anticoagulantwww.CLOT-ED.com 44
  45. 45. Tests for Fibrinogen Quantitative Fibrinogen Thrombin Time (TT) Measures the amount of Assesses the functionality of fibrinogen present in plasma fibrinogen in plasma Low levels, termed TT clotting time prolonged hypofibrinogenemia, can be – HEPARIN inherited but generally are due – Direct thrombin inhibitors to acquired causes such as – Hypofibrinogenemia DIC, liver disease, or – Dysfibrinogenemia fibrinolytic therapy – Elevated fibrin split products High levels are seen in inflammatory states since Time for clot formation ~ 15 seconds fibrinogen is an acute phase reactant Incubate at 37 oC for ~2 minutes 0.2 ml Diluted Thrombin 0.2 ml Plasmawww.CLOT-ED.com 45
  46. 46. Quantitative Fibrinogen Calibrator plasma is serially diluted Time for clot formation (1:5, 1:10, 1:20, and 1:40) to establish ~ 5-14 seconds a reference curve (see graph below) Patient plasma is diluted 10 fold (1:10) in buffer Incubate at 37 oC for ~3 minutes 0.1 ml Thrombin 0.2 ml Diluted Plasma 100 Patient clotting time in seconds is Clotting Time-Seconds 35.0 read against the reference curve 18.3 − Patient clotting time of 8.0 9.5 seconds equates to 281 mg/dl 8.0 5.1 Patient fibrinogen in example at right Fibrinogen concentration is inversely 1 proportional to clotting time 1 10 10 58 115 100 230 281 460 1000 mg/dl Fibrinogenwww.CLOT-ED.com 46
  47. 47. Coagulation: A Balancing Act Generate Inhibit Thrombin Thrombin Formationwww.CLOT-ED.com 47
  48. 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 Inhibitorswww.CLOT-ED.com 48
  49. 49. Fibrinolysiswww.CLOT-ED.com 49
  50. 50. Hemostasis: A Delicate Balance Generates Generates Thrombin Plasmin Form a Dissolve a Thrombus Thrombuswww.CLOT-ED.com 50
  51. 51. Fibrinolytic System TAFI Thrombin TM Thrombin TAFIa TM PC APC ‡ Fibrinogen FIBRIN tPA Plasminogen FXIII Plasmin PAI-1 ‡ Fibrin(ogen) Degradation Products Antiplasminwww.CLOT-ED.com 51
  52. 52. Breakdown of Fibrin(ogen) FPB FPB FPA & B FPA & B Fibrinogen Fibrinogen Fibrin Thrombin Fibrin Monomer Clot Fibrin Monomer Fragment X Fragment X Fibrin Polymer Fibrin Polymer D D Y Y FXIII FXIIIa PLASMIN D D E E D D D D E E D D D D E E D D D-dimer D D D D E E D D D D E E D Dwww.CLOT-ED.com 52
  53. 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 α2-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)www.CLOT-ED.com 53
  54. 54. Summarieswww.CLOT-ED.com 54
  55. 55. Time Frame for Hemostasis Platelets Coagulation Factors Fibrinolytic Proteins Primary Primary Secondary Secondary Fibrinolysis Fibrinolysis Hemostasis Hemostasis Hemostasis Hemostasis •• Vessel constriction Vessel constriction •• Activation of Activation of •• Activation of Activation of occurs immediately occurs immediately coagulation coagulation fibrinolytic fibrinolytic factors occurs in factors occurs in proteins happens proteins happens •• Platelet adhesion Platelet adhesion seconds seconds immediately immediately occurs in seconds occurs in seconds •• Fibrin forms in Fibrin forms in •• Dissolving the Dissolving the •• Platelet aggregation Platelet aggregation minutes minutes thrombus thrombus takes minutes takes minutes requires hours requires hourswww.CLOT-ED.com 55
  56. 56. Bleeding: Balance is Disrupted Presence of Inhibitors*•Pharmacologic (warfarin, heparin)•Allo or Auto-antibodies to Factorswww.CLOT-ED.com 56
  57. 57. Thrombosis: Balance is Disrupted Inhibitors* • Activated Protein C • Protein S • Antithrombinwww.CLOT-ED.com 57
  58. 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 hemostasiswww.CLOT-ED.com 58
  59. 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, 1996. Crowther MA, et al. Practical aspects of anticoagulant therapy (Chapter 89). In: Colman RW, ed. Hemostasis and Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001. 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):768-81. Greaves M, Preston FE. Approach to the bleeding patient (Chapter 48). In: Colman RW, ed. Hemostasis and Thrombosis, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001. 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, 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006. Link KP. The discovery of Dicumarol and its sequels. Circulation 1959;19(1):97-107. 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):782-98. Physicians’ Desk Reference, 60th ed. Montvale: Thomson PDR, 2006. 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, 2000. 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, 2006. van den Besselaar, AMHP, Gralnick HR, Lewis SM, Editors. Thromboplastin Calibration and Oral Anticoagulant Control. Dordrecht: Martinus Nijhoff Publishers, 1984.www.CLOT-ED.com 59