Coagulation Simplified… Published by ACKNOWLEDGEMENTS CONTENTS We gratefully acknowledge the support and funding provided by the Ontario Ministry of Health 1. The Basics of Coagulation and Clot Breakdown . 4–7 and Long-Term Care. 2. Routine Coagulation Tests . 8–17 Special thanks to the following people and organizations who provided their expertise in Evaluating coagulation in the laboratory . 8 reviewing the content of this handbook: Sample collection for coagulation testing . 9 Prothrombin Time (PT) . 10 L Gini Bourner (QMP-LS Hematology Committee) International Normalized Ratio (INR) . 11 L Dr. Jeannie Callum Activated Partial Thromboplastin Time (APTT) . 12 L Dr. Allison Collins Thrombin Time (TT) . 13 Fibrinogen . 14 L Dr. William Geerts D-dimer . 15 L Dr. Alejandro Lazo-Langner Anti-Xa assay . 16 L Dr. Ruth Padmore (QMP-LS Hematology Committee) Summary . 17 L Anne Raby (QMP-LS Hematology Committee) 3. Anticoagulant Drugs . 18–25 L Dr. Margaret Rand Unfractionated Hepari n (UFH) . 18 L Dr. Alan Tinmouth Low Molecular Weight Heparins (LMWHs) . 19 Fondaparinux . 20 Warfarin . 21 Thanks also to: Direct Thrombin Inhibitors (DTI) . 23 L Dale Roddick, photographer, Sunnybrook Health Sciences Centre Direct Xa Inhibitors . 25 L Reena Manohar, graphic artist, Sunnybrook Health Sciences Centre 4. Evaluating Abnormal Coagulation Tests . 26–29 L The ECAT Foundation Prolonged PT / INR with normal APTT . 26 CLOT-ED Images used or modified with permission from Prolonged APTT with normal PT / INR . 27 the ECAT Foundation, The Netherlands. Prolonged APTT and PT / INR . 28 Prolonged Thrombin Time (TT) with normal or prolonged APTT and PT / INR . 29 March 2013 5. Approach to the Evaluation of the Bleeding Patient . 30–35 General Disclaimer: 6. Diagnosis and Emergency Management of While the adv ice and information in this handbook are believed to be true and accurate at Common Bleeding Disorders . 36–45 the time of publishing, neither the authors nor the publishers accept any legal responsibility von Willebrand disease (VWD) . 36 or liability for any errors or omissions in the information provided, or for any of the Disorders of platelet function . 38 recommendations made. Any decision involving patient care must be based on the judgement Hemophilia A and B (Factor VIII and IX deficiency) . 40 of the attending physician according to the needs and condition of each individual patient. Factor XI deficiency . 43 Key References . 45 2 3 1. THE BASICS OF COAGULATION AND CLOT BREAKDOWN Lesley Black & Rita Selby Breakdown Hemostasis is a complex process in which multiple components of the blood clotting system 2. Secondary hemostasis = activation of coagulation factors and generation Clot & Coagulation 1. are activated in response to vessel injury to control bleeding. of thrombin: Hemostasis is composed of four major events: Initiation of coagulation 1. Primary hemostasis L Tissue factor (TF) is released from injured tissue cells, endothelial cells and monocytes. 2. Secondary hemostasis L TF and Factor VIIa form the TF / Factor VIIa complex. 3. Fibrin clot formation and stabilization L TF / Factor VIIa activates a small amount of Factor IX and X to generate a small amount 4. Inhibition of coagulation of thrombin. L Factor XII (and other “contact” factors) play a minor role in the activation of Factor XI. 1. Primary hemostasis = vasoconstriction and platelet plug formation: L The key component of primary hemostasis is the platelet. Amplification phase L Primary hemostasis is triggered by injury to the vessel wall, exposing subendothelial L Thrombi n activates Factor V to Va, Factor VIII to VIIIa and activates more platelets. collagen. L Thrombin also activates FXI to FXIa. L Vasoconstriction occurs at the site of injury to reduce blood flow. L Adhesion: von Willebrand factor GPllb / llla Propagation phase adheres platelets to exposed L Additional Factor Xa is produced when TF / Factor VIIa complex activates Factor IX. The subendothelial collagen via the Dense granules platelet receptor glycoprotein Ib Platelet resultant Factor IXa along with Factor VIIIa forms the tenase complex which then converts (GPIb) . Platelets also adhere more Factor X to Xa. directly to collagen by other Alpha granules L Factor Xa and Va along with calcium and a phospholipid (PL) surface (act ivated platelets) receptors. GPlb / IX Endothelium form the prothrombinase complex which converts prothrombin (Factor II) to large amounts Collagen VWF Injury site L Aggregation: Platelets aggregate collagen exposed of thrombin (Factor IIa). with each other with the help of fibrinogen that binds to activated glycoprotein IIb-IIIa (GPIIb / IIIa), 3. Fibrin clot formation and stabilization: forming a platelet plug. Platelet L Thrombin converts fibrinogen to fibrin monomers which polymerize to form a soluble clot. aggregates also provide the Thrombin then activates Factor XIII which cross-links the fibrin monomers and stabilizes phospholipid surface necessary the clot. for coagulation factor activation. GPllb / IIIa Activated Fibrinogen Platelet Collagen GPlb / IX VWF 4 5 1. THE BASICS OF COAGULATION AND CLOT BREAKDOWN Breakdown Secondary hemostasis, fibrin clot formation and stabilization: 4. Inhibition of coagulation = inhibition of thrombin generation and fibrin Clot & Coagulation 1. clot breakdown (fibrinolysis) Contact Factors (HMWK, Prekallikrein) Inhibition of thrombin generation INITIATION L At the same time that a clot is being formed, the clotting process also starts to shut itself FXII FXIIa Tissue off to limit the extent of the thrombus formed. Factor PROPAGATION FVIIa L Thrombin binds to the membrane receptor thrombomodulin and activates Pro tein C to FXI FXIa Activated Protein C (APC). 2+ TF - VIIa complex Ca L APC combines with its cofactor Protein S which then inhibits Factors Va and VIIIa, slowing FIX FIXa down the coagulation process. 2+ FVIII FVIIIa Ca +PL L Thrombin bound to thrombomodulin becomes inactive and can no longer activate procoagulant factors or platelets. FX FXa 2+ L The endogenous anticoagulant, antithrombin inhibits the activity of thrombin as well FV FVa Ca +PL as several of the other activated factors, primari ly Factor Xa. Prothrombin Thrombin (FII) (FIIa) Fibrinolysis AMPLIFICATION L Tissue plasminogen activator (t-PA) converts plasminogen to plasmin which breaks down cross-linked fibrin to several fibrin degradation products, the smallest of which is D-dimer. Fibrinogen Soluble Fibrin L Thrombin activatable fibrinolysis inhibitor (TAFI) prevents the formation of plasmin. L Anti-plasmin and plasminogen activator inhibitor-1 (PAI-1) inhibit plasmin and t-PA Fibrin Clot Formation FXIIIa respectively. and Stabilization Insoluble Plasminogen Antiplasmin TAFI cross-linked !brin tPA PAI - 1 Plasmin Fibrin(ogen) Fibrin Clot Degradation Products D-dimers 6 7 2. ROUTINE COAGULATION TESTS Elena Brnjac & Rita Selby Sodium Citrate Tube Evaluating coagulation in the laboratory Sample collection for coagulation testing (Blue Top) L In the coagulation laboratory, the coagulation factors are divided into: L To assess coagulation “in vitro,” the laboratory measures the time • Extrinsic pathway factors (Factor VII) taken to form a clot. • Intrinsic pathway factors (Factors XII, XI, IX, VIII) L Blood is collected into a blue top tube containing sodium citrate anticoagulant (which chelates calcium) to prevent blood clotting • Common Pathway factors (Factors X, V, II, Fibrinogen) in the tube during transport. 2. Routine Routine 2. ATTENTION Tests Coagulation L Memorizing which factors belong to the extrinsic, intrinsic and common pathways Sodium Citrate Tube Coagulation testing respective ly will make evaluating the causes of abnormal coagulation tests easier. (Blue Top) MUST only be sent in a sodium citrate Centrifugation (blue top) tube. Intrinsic Pathway Extrinsic Pathway ! XII XI VII IX Tissue Factor L Plasma (the liquid component of blood that contains the clotting VIII factors) is then separated from the platelets (phospholipid source) APTT X PT Intrinsic Extrinsic by centrifugation. + Common V Pathway + L Common II Common Later we will see how adding back phospholipids and calcium is important in standardizing routine coagulation tests. Fibrinogen Fibrin Clot L Some common problems that may result in spurious coagulation Plasma test results are: Buy Coat • Blood collected into incorrect type of tube (not a sodium citrate Red L Here is another picture to help with memorizing the coagulation cascade without the tube) Cells Roman numerals: • Incorrect plasma to citrate ratio (e.g. underfilling of tube or 8 5 patient’s hematocrit > 0.55 L/L) 12 11 9 10 2 1 • Heparin contamination of sample (e.g. incorrect order of sample Thrombin Fibrinogen PTT collection or sample collected from central lines) • Clotting in tube from traumatic venipuncture or inadequate 7 mixing INR • Hemodilution of sample • The common pathway factors can be memorized by thinking of the denominations of dollars in Canada: factors 10, 5, 2 and 1 • The PT/INR pathway starts with factor 7 and includes the common pathway factors • The APTT pathway starts from the left at factor 12, counts backwards to factor 8 (skipping factor 10) and includes the common pathway factors 8 9 2. ROUTINE COAGULATION TESTS Prothrombin Time (PT) International Normalized Ratio (INR) L The PT is used to assess deficiencies or Extrinsic L The International Normalized Ratio (INR) was developed to standardize the PT to allow inhibitors of the extrinsic pathway Pathway for monitoring of oral vitamin K antagonist therapy