Hematology - 13 Secondary Hemostasis

HEMATOLOGY SECONDARY HEMOSTASIS SECONDARY HEMOSTASIS/BLOOD COAGULATION • OUTLINE • Introduction • • Secondary Hemostasis o Coagulation Factors o Pathways of Coagulation Factors o Bleeding Disorders • Laboratory Evaluation of Secondary Hemostasis o Coagulation Time o Plasma Recalcification Time o Partial Thromboplastin Time o Activated Partial Thromboplastin Time o Prothrombin Time o Other Tests o Mixing Studies/ Substitution Test o International Normalized Ratio Supplemental Notes o Stages in Blood Coagulation: Waterfall or Cascade Theory o Classification of Coagulation Factors according to Function o Classification according to Physical Properties o Coagulation Testing Requirements o Blood Preparation Used in Coagulation Tests o Factor VIII and wWF o Factor XI & Contact Factors o Thrombin o Fibrin Structure and Fibrin Formation, Factor XIII o Coagulation System o Hemostatic System o Coagulation Regulatory System INTRODUCTION • • • • • • This mechanism deals with the activation of a series of coagulation proteins in the plasma. Activated coagulation protein factors are referred to as SERINE PROTEASES ( Factor VII -> Factor VIIa), which are with subscript a. This mechanism culminate in the formation of stable fibrin clot (in vivo). In laboratory tests, which mimics the mechanism results in the fibrin polymer. Although the vascular intima and platelets are associated with primary hemostasis, all systems interact, including fibrinolysis in early and late hemostatic events. Example of this is, platelets, although a key component of primary hemostasis, also secrete coagulation factors stored in their granules and provide an essential cell membrane phospholipid (PF3) on which coagulation complexes form. Blood coagulation is a sequential process where the product of an interaction of serine proteases with cofactors, collagen and other coagulation factors, etc. will affect the formation of the next complex and so on. Knowledge of the secondary hemostatic mechanism leads to understanding of the tests in the laboratory, very useful to the physician for treatment and management of disorders. Many tests are available for use, the most widely used are partial thromboplastin time (PTT/APTT), prothrombin time (PT), thrombin time, quantitative fibrinogen concentration assay, INR, CLAUSS procedure, factor identification tests, Stypven time, reptilase time, etc. These ensures correct diagnosis and management of disorder of patient. Like in any testing, coagulation tests are sensitive to the effects of too much coagulation deviation in temperature, pH and other factors. Testing needs strict adherence to requirements and protocols so that results are reliable. The patient are the ultimate beneficiaries when the laboratorian/medical technologist come up with results of quality. • • Involves the activation of plasma proteins (serine proteases) in the coagulation system to form a fibrin meshwork. o Meshwork occurs in platelet plug Focuses on coagulation and coagulation factors o Platelets are involved due to Platelet factor (PF) 3 Coagulation - A process whereby, on vessel injury, plasma proteins, tissue factors, and calcium interact on the surface of platelets to form a stable fibrin clot. Platelets also interact with fibrin to form a stable platelet-fibrin clot. o Interplay of various factors o In vivo - stable fibrin (endpoint of circulation o In vitro - fibrin polymer (end point of assays) COAGULATION FACTORS Roman Preferred Descriptive I Fibrinogen II Thrombin III Tissue factor IV Calcium V Proaccelerin VI Omitted VII Proconvertin VIII IX X Xi XII XIII • • • • Synonyms Biochemistry Ia-Fibrin IIa-Thrombin Thrombloplastin Glycoprotein Labile factor Lipoprotein Metal ion Glycoprotein Stable factor, Serum Glycoprotein prothrombin conversion accelerator Antihemophilic Antihemophilic Factor (AHF) Globulin (AHG), Antihemolytic factor A Plasma Antihemophilic factor Thromboplastin B/Christmas factor Component (PTC) Stuart-Prower Stuart factor Factor Prower factor Plasma Antihemophilic factor Beta/Gamma Thromboplastin C globulin Antecedent (PTA) Hageman Contact factor Sialoglycoprotein Factor Glass factor Fibrin Laki-Lorand Factor Beta/Gamma Stabilization Fibrinase Globulin Factor Prekalikrein Fletcher factor Plasma protein High Molecular Fitzgerald/William/ Weight Flaujeac factor Kininogen (HMWK) Use Arabic VI - omitted because it is activated factor V Thrombin - from prothrombin (a = activated) EDTA & citrate - chelates calcium; inactivation of Ca o EDTA - routine hema o Citrate - coagulation testing o CLASSIFICATIONS OF COAGULATION FACTORS • • According to hemostatic function: o Zymogens ▪ Inactivated form of the coagulation factor. ▪ Proenzyme ▪ Prothrombin (Factor IIa) o Cofactors ▪ Substances needed by the enzyme to proceed with the reaction. ▪ Aka accelerator ▪ Factor III, V, VIII, HMWK, Tissue Factor o Substrates ▪ Factor I o Enzymes/Serine Protease ▪ Activated form of the coagulation factor. o Transamidase/ Transglutaminase ▪ Factor XIII According to Physical properties o Prothrombin group/Vit. K dependent group ▪ Requires Vitamin K for its production in the liver. ▪ Prothrombin (Factor II) - main proenzyme ▪ Vit. K deficiency will cause decreased synthesis ▪ Factors II, VII, IX, X ▪ Protein C, Protein S - regulatory proteins; are not CF, they are potent anticoagulant but classified with prothrombin group ▪ Present in both serum in plasma ▪ Not consumed during coagulation ▪ Can be adsorbed by barium sulfate/aluminum hydroxide ▪ Not present in centrifugated/adsorbed plasma ▪ Coumarin (oral anticoagulants) - compete with prothrombin group in synthesis in the liver o Fibrinogen group/Labile group ▪ Factors consumed during the clotting process ▪ Factors I, V, VII, XIII ▪ Do not require Vit. K for synthesis ▪ Consumed during coagulation • Absent in serum, present in plasma ▪ Not adsorbed by barium sulfate/aluminum hydroxide ▪ Present in adsorbed plasma ▪ Increased in inflammatory conditions ▪ Fibrinogen - acute phase protein, increases in surgery to initiate stabilization of fibrin clot for addressing blood clots o Contact group ▪ First factors that are activated during the coagulation process. (Intrinsic pathway) ▪ XI, XII, Prekalikrein, HMWK ▪ Not consumed in coagulation ▪ Independent from vit. K for synthesis ▪ Not adsorbed by barium sulfate/aluminum hydroxide ▪ Very stable ▪ Active in activation of plasminogen and fibrinolysis • • • • • • • • Activity will occur in platelet plug itself by secondary hemostasis in a sequential manner All pathways occur at the same time and converge in common pathway Waterfall cascade theory Both pathways occur simultaneously Extrinsic - more prominent o Named because most factors found intrinsically o Routinely tested by APTT ▪ Same with PTT, different in ref. values and has activator (shortens time, hastens activity) Converges/merges in common pathway to activate 10 to 10a to start common pathway o Conversion of prothrombin to thrombin for the conversion of fibrinogen to fibrin for the formation of a stable fibrin clot EXTRINSIC PATHWAY • • • • • PATHWAYS OF COAGULATION FACTORS • Intrinsic pathway o Activated by contact with S.E.C. o More active and initiates coagulation Extrinsic pathway o Activated by contact with Tissue thromboplastin Common pathway o A cascade that results in the activation of prothrombin that is needed to convert fibrinogen to fibrin. • Composed of Factor VII. Activated by the release of Tissue factor from the injured vessel into the plasma. Tissue factor – Also known as tissue thromboplastin; Secreted by tissues. Tissue factor is capable of binding to Factor VII, converting it to Factor VIIa. Factor VIIa will then activate Factor X to Factor Xa. INTRINSIC PATHWAY • • • • Composed of Factors VIII, IX, XI, XII, HMWK and Prekallikrein. Activated when the S.E.C. comes into contact with the coagulation factors. Factors XI, XII, Prekallikrein, and HMWK interact on the surface to activate Factor IX to Factor XIa Factor Xia will then react with Factor VIII, PF3, and Calcium to activate Factor X to Factor Xa. BLEEDING DISORDERS (C.F. DEFICIENCIES) Coagulation Factor Deficiency Factor I Factor II Factor V Factor VII Factor VIII Factor IX • • • • • HMWK - cofactor that increases conversion of prekalikrein to kalekrein (activates XII to XIIa) XIIa converts XI to Xia XIa - first factor to require calcium in lymph PF3 - phospholipid; important in platelet factors Negatively charged surfaces are capable of activating Factor XII. o In vivo – Collagen, Elastin ▪ siliconized/plastic container o In vitro – Glass, Kaolin, Ellagic acid (contact surface) Clinical Significance Afibrinogenemia Hypofibrinogenemia Dysfibrinogenemia Prothrombin deficiency Hypoprothrombinemia Owren’s Disease Parahemophilia Hypoproconvertinemia Hemophilia A Classic Hemophilia Royal’s disease Christmas factor disease HemoPhilia B Factor X Stuart-Prower disease Factor XI HemoPhilia C Rosenthal Diseas Factor XIII Associated with Poor wound healing • No person with fibrinogenemia o <200 - hypo o >500 - hyper o Dys - dysfunctional; present fibrinogen but do not function well; due to change in amino acid LABORATORY EVALUATION OF SECONDARY HEMOSTASIS COMMON PATHWAY • • • • Starts with the activation of Factor X to Factor Xa by either intrinsic or extrinsic pathway. Factor Xa, with the help of Calcium, Factor V, and PF3, will activate Prothrombin to Thrombin. Thrombin will then activate Fibrinogen to become Fibrin. (Fibrin monomer) Upon the action of Factor XIII, Fibrin monomers will be strengthened and produce a stable fibrin clot. • • Platelet aggregometer - reference method COAGULATION TIME • • • • • • • • Factor Xa requires Ca, V (cofactor), PF3 to convert II to IIa (prothrombin to thrombin) Prothrombinase complex - conversion of II to IIa Thrombin - main role is to convert fibrinogen to fibrin o Also converts XIII to XIIIa for the stabilization of fibrin to produce stable fibrin clot • • Aka clotting time Tests the composite action of all plasma factors acting simultaneously. Measure both pathways Not sensitive, requires numerous conditions for positive Clotting time is a measure of the ability of the blood to clot and is not influenced by the platelet functions other than PF3. o The time between drop of blood to first observable fibrin thread o Observed for fibrin thread o Normal value: 2-4 mins It also measures only the time required for the formation of the traces of thrombin sufficient to produce a visible clot. Rarely used • • • Micro Methods o Slide or Drop Method 1. Puncture the ring/middle finger 2. Start timer then transfer blood to slide every 30 seconds. 3. Observe for fibrin string by lifting lancet. o Capillary or Dale and Laidlaw’s Method 1. Collect non-heparinized blood 2. Start timer and fill 3 capillets 3. After 30 secs, break the capillet 4. Observe for fibrin string. Macro Methods o More accurate o Superior for there is less contamination of the plasma with tissue fluids when blood is drawn from a vein. o Lee-White Method or Whole Blood Clotting Time ▪ Uses at least 3 tubes 1. Wash the test tube using sterile NSS 2. Collect 3 ml of blood and transfer it to the test tube 3. Every 30 secs, tilt the tube and observe for clot. Normal Value: 5-10 minutes • • • OTHER TESTS • • PLASMA RECALCIFICATION TIME • • • • • Recalcification - coagulation of plasma More sensitive method than the coagulation time of whole blood because there is an activator added. May reveal abnormality which is not detectable by the determination of the clotting time of venous blood. The activated recalcification time makes use of 0.025 M CaCl2 as Normal Value: Less than 50 seconds • PARTIAL THROMBOPLASTIN TIME • • • • Test for the intrinsic and common pathways of coagulation. Calcium ions and phospholipids that substitute for platelet phospholipids are added to blood plasma. Detects deficiencies in Factors XII, XI, IX, VIII Normal value: 70 - 100 seconds • • • • • • • Similar to PTT test but with the addition of activators to hasten the production of a visible result. o silica or ellagic acid, kaolin, and phospholipids activators Test for the deficiencies of factors in the intrinsic system. Uses patient’s plasma or platelet-poor plasma Time between addition of calcium and first observable clot o Machines may use digital probe; change in optical density, light passing through o Automated techniques are based on manual principle o Calcium is added because it is used up Reagent: phospholipids or substitute Normal Range: 25-35 seconds Phospholipid deficiency PROTHROMBIN TIME • • Measures the extrinsic and common pathway of coagulation. It is used to monitor oral anticoagulant therapy. (coumarol) Thrombin time o Measures the time of the conversion of fibrinogen to fibrin clot. o For the functionality of fibrinogen o Patient’s lasma + thrombin o Normal range: 10-20 seconds Reptilase time o Evaluation of a prolonged thrombin time (TT): It is mainly used to confirm or exclude the presence of heparin in the specimen or specimen type. o Uses Reptilase from the snake venom of Bothrops atrox. o Normal Range: 18-20secs Stypven Time (Rusell’s viper venom Time) o Used to distinguish deficiencies of Factor X and those of Factor VII. It is also used to detect deficiencies in prothrombin, fibrinogen and factors V and X. It differs from prothrombin time in that deficiencies in factor VII are not detected. Duckert’s Test o Used to detect deficiency in Factor XIII. MIXING STUDIES/SUBSTITUTION TEST • • ACTIVATED PARTIAL THROMBOPLASTIN TIME • Used to detect VII deficiency. Principle: o Tissue thromboplastin and calcium added to plasma react to fibrinogen to form a clot. The thromboplastin added to the plasma takes the place of the tissue juice formation of extrinsic thromboplasitn. The prothrombin time is therefore prolonged if there is a deficiency of Factors V, VII or X or a very severe deficiency of Factor I and II. o Complete thromboplastin o Reagent: from brain, test animal, ▪ Brain - rich in phospholipids Normal Range: 10-14 seconds • • A test used to pinpoint the exact coagulation factor that is deficient. Principle: o The patient’s plasma is mixed with a plasma/serum that contains known coagulation factors and PT and aPTT tests are repeated. ▪ If one or both of the results become normal, a coagulation factor deficiency is suggested. ▪ If the results remain the same, there might be a presence of an inhibitor eg. Warfarin, that inhibits the cascade. Reagents Does not contain Contains Fresh Plasma All Coagulation Factors Aged Plasma V, VIII I, II, VII, IX, X, XI, XI Absorbed Plasma II, VII, IX, X I, V, VIII, XI, XII Fresh Serum I, V, VIII, XIII II, VII, IX, X, XI, XII Aged Serum I, II, V, VIII, XIII VII, IX, X, XI, XII Sample Pathway Coagulation Factors Present Extrinsic VII Intrinsic VIII, XI, IX, XII Common I, II, V, X • • • Example 1 o A patient yielded the following results: ▪ aPPT – 45 secs ▪ PT – 10.3 secs o The medical technologies then made 2 aliquots from the sample. The first aliquot was mixed with Aged serum while the second aliquot was mixed with Adsorbed plasma. The tests are repeated and yielded the following results: ▪ Aged serum – Not Corrected ▪ Adsorbed plasma – Corrected o Using the following results, identify which coagulation factor is deficient. o PT is normal so it means that Factor VII should be excluded from the suspected factor deficiency/ies o aPTT is prolonged so it is tested on both aliquots. o Suspected factor deficiencies: Factors 8, 9, 11, 12 o Aged serum lacks: Factors 1, 2, 5, 8 –Not corrected o Adsorbed plasma lacks: 2, 7, 9, 10 –Corrected o Deficient Factor: VIII(8) Example 2 o A patient yielded the following results: ▪ aPPT – 48.5 secs ▪ PT – 19.3 secs o The medical technologies then made 2 aliquots from the sample. The first aliquot was mixed with Aged serum while the second aliquot was mixed with Adsorbed plasma. The tests are repeated and yielded the following results: ▪ Aged serum – Corrected ▪ Adsorbed plasma – Not Corrected o Both PT and aPTT are prolonged so they are both repeated. o Suspected factor deficiencies: Factors 1, 2, 5, 10 o Aged serum lacks: Factors 1, 2, 5, 8 –Corrected o Adsorbed plasma lacks: 2, 7, 9, 10 –Not corrected o Deficient Factors: II(2) and X(10) Summary • INTERNATIONAL NORMALIZED RATIO (INR) • • • • • • Modification of Prothrombin time o Due to labile characteristics Uses perfect complete thromboplastin housed in Geneva Not reported in seconds Computed by prothrombin / mean For monitoring oral anticoagulant therapy International Sensitivity Index (ISI) SUPPLEMENTAL NOTES STAGES IN BLOOD COAGULATION: WATERFALL OR CASCADE THEORY • • • • Contact Phase Activation of the Common Pathway Conversion of Prothrombin to Thrombin Formation of the Stable Fibrin Clot CLASSIFICATION OF COAGULATION FACTORS ACCORDING TO FUNCTION • • • • Serine proteases- activated form; enzymes; denoted with subscript small letter “ a” o Factors IIa, VIIa, IXa, Xa, Xia, XIIa Cofactors/ Accelerators- hastens the coagulation factors’ activity o Factor III, Factor V, Factor VIII, HMWK Substrate- acted by enzymes/ serine proteases o Factor I ( fibrinogen) Transamidase/ Transglutaminase o Factor XIII CLASSIFICATION OF COAGULATION FACTORS ACCORDING TO PHYSICAL PROPERTIES • Prothrombin or Vitamin K –Dependent Group o Composed of Factors II, VII, IX, X and the regulatory proteins, Protein C and its cofactor Protein S. also protein Z. They all have structural resemblance to prothrombin. Protein S and Protein Z are both cofactors. o Requires Vitamin K during synthesis , as it catalyzes an essential posttranslational modification of this group of factors o In the presence of Coumadin/coumarin/ warfarin/dicumarol is therapeutic inhibitor of Vitamin K, the Vit-K dependent procoagulants are released from the liver without the second carboxyl group, and so they cannot bind to calcium and PPl so they cannot participate in the coagulation reaction. Vit K antagonism is the basis for oral anticoagulant of the aforementioned drugs. o Essential for the assembly of three membrane complexes leading to the generation of thrombin. Each complex is is composed of a Vit-K dependent serine protease, its nonenzyme cofactor, and calcium, bound to the negatively charged to the PPL membranes of activated platelets or tissue-factor bearing cells. o Extrinsic tenase - composed of factors VII and tissue, respectivelye factor, and it activates Factors IX and X, which are components of the next two complexes, intrinsic tenase and prothrombinase. o Intrinsic tenase -composed of factor IXa and its cofactor, VIIIa, it also activates factor X but much more efficiently than the TF: VIIa complex. o Prothrombinase - composed of factor Xa and its cofactor Va; this converts prothrombin to thrombin in a multistep hydrolytic process that releases thrombin and a peptide fragment called ptothrombin fragment 1.2 ( F1.2). Prothrombin fragment 1.2 in plasma is thus a marker for thrombin generation. • • Fibrinogen or thrombin-sensitive group - composed of factors I. V. VIII.XIII. these factors are consumed during coagulation. Vitamin K is not necessary for their synthesis. They are absent in serum but present in plasma. They are not adsorbed by Al(OH)3 or BaSO4. They are increased in pregnancy and in inflammatory conditions. They are also increased as a result of use of oral contaceptives.Factors V and VIII are known to decrease during blood storage in vitro. Contact group -composed of factors XI, XII, Prekallikrein and High molecular weight kininogen( HMWK). They are involved in the intrinsic coagulation pathway. They are moderately stable and are not consumed during coagulation.. They are not dependent on Vitamin K for their synthesis. Prekallikrein and HMWK also function in the activation of plasminogen( in the fibrinolytic system). • • • COAGULATION TESTING REQUIREMENTS • • • • • • • • • • • Clean venipuncture- avoid admixture with tissue juice because this will cause erroneously short results. Preferred anticoagulant – 0.109 M ( or 3.2% ) buffered sodium citrate and is with 1:10 dilution o ( 1 part anticoagulant plus 9 parts blood) Blood should be collected in second tube( first should be “ discard” tube) to ensure no admixture with tissue juice In phlebotomy, release the tourniquet as soon as blood enters the first tube or syringe. All tubes for coagulation studies should be “ non-contact” surface, meaning plastic and siliconized container Blood specimens with a haematocrit of 55% and more contains less plasma in relation to the total volume of blood in the tube. It is necessary to compute for the amount of sodium citrate (the anticoagulant) using the formula: o Amount of sodium citrate = 100- haematocrit/ 595hematocrit X amount of blood used Specimen should be checked for clots prior to testing. Specimen should be centrifuged within 1 hour of obtaining the sample. After centrifugation, blood should be removed immediately with a plastic or siliconized pipette and stored to a plastic or siliconized container. Only the ¾ of the plasma layer should be aspirated.. For some tests, centrifugation of the sample at a temperature of 24degrees Centigrade is advisable.For this process, a refrigerated centrifuge is required. The buffering effect of red cells is lost once the sam at ple is centriguged and the plasma exposed to air. Testing should be done immediatelt on centrifuged samples or the plasma should be stored at 4 degrees Centigrade for a time not to exceed 2 hours. Pipettes, test tubes, and othr materials which come in contact with plasma should have a “ non-contact” surface ( plastic, siliconized) Specimens should be stoppered always. Specimens should not be frozen if testing can be done within 2 hours after collection. If freezing is necessary, it should be done rapidly at -20 degrees Centigrade or lower. With slow freezing,ice particles form that may denature the clotting proteins. If frozen properly,fibrinogen is stable for at least 4 hours after thawing and survives refreezing and thawing. • • With the exception of platelet function tests,plasma for coagulation testing should be platelet poor( less than 10X 10 to the 9th/ L). Avoid hemolyzed plasma since results will not be dependable. Red cells contain ADP which is released in the plasma may activate the platelets. Also, automated machines may not be able to detect the end point on some lipemic or icteric plasma samples. For storage of plasma: o platelet-poor plasma(ppp) or ordinary plasma- stored at -40 degrees Centigrade or lower for at least several week without loss of most factors. The plasma should initially be frozen( in covered plastic containers)nvery quickly using liquid nitrogen or a temperature ofn-80 degrees Centigrade to -40 degrees Centigrade. o When thawing frozen plasma, it should be done rapidly in a 37degrees Centigrade incubator or water bath. Remove the plasma from the incubator as soon as it is thawed. Thawed plasma cannot be refrozen. This is the reason why plasma should be stored in aliquots. Changes in the pH samples can affect values by causing prolongation of clotting time. Changes in Ph are mediated by the loss of CO2 from the sample. As CO2nisn lost, the pH of the sample increases. The buffered citrate solution contained in evacuated tubes protect samples against such loss for a period of time. Red cells if not exposed to air have a buffer effect also. Normal samples collected collected in evacuated tubes and stored unopened at room temperature for as long as 6 hours show no significant changes in PT or APTT results. If samples are left at room temperature for an extended time, factors V and VIII are likely to deteriorate. Factors VII and XI tend to be prematurely activated at refrigerator temperature( 4 degrees Centigrade). BLOOD PREPARATION USED IN COAGULATION TESTS • • • • • Patient’s plasma: 1. Mix 4.5 mL blood and 0.5 mL 0.109 M sodium citrate 2. Centrifuge at 1500 rpm for 5 minutes Adsorbed normal plasma: o Adsorption – process of removing factors II, VII, IX and X from normal plasma bythe use of certain insoluble salts of alkaline earth like aluminium hydroxide gel, barium sulphate and carbon phosphate. Factors XII,V and VIII are not adsorbed. 1. Mix 2 mL of plasma and add 0.2 mL adsorbing reagent. 2. Centriguge at 3,000 rpm for 3 minutes. 3. Refrigerated plasma should be used within 2 hours. Aged normal plasma: 1. Collect plasma in the usual manner. 2. Incubate at 37 degrees Centigrade for 24 hours 3. Store in aliquots at 20 degrees Centigrade. Aged normal serum: 1. Collect serum in the usual manner. 2. Allow to stand at room temperature for 24 hours. 3. Divide in aliquots and freeze. Platelet-poor plasma( ppp) 1. Mix 9 parts of blood and 1 part of 0.109 M sodium citrate 2. Centrifuge at 3,000 for 30 minutes • • Platelet-rich plasma 1. Mix blood as in letter e. 2. Centrifuge at 1,500 rpm for 5 minutes Significant Platelet Levels: 1. Less than 100,000/uL= abnoemally low 2. 30,000-50,000= bleeding possible with trauma 3. Less than 30,000/uL=spontaneous bleeding possible 4. Less than 5,000/uL=severe spontaneous bleeding • FACTOR VIII AND VON WILLEBRAND FACTOR (VWF) • • Factor VIII is unstable in plasma, it circulates bound to vWF. During coagulation, thrombin cleaves factor VIII from vWF and activates it. Factor VIIIa binds to activated platelets and forms the intrinsic tenase complex with factor IXa and calcium. Like factor Va, factor VIIIa is also inactivated by Protein C. o Factor VIII deteriorates more rapidly than the other coagulation factors in sored blood. In thawed component plasma,, factor VIII level drops to approximately 50% after 5 days. vWF is a large multimeric glycoprotein that participates in platelet adhesion and transports the procoagulant protein factor VIII. vWF is composed of multiple subunits of 240,000 Daltons each. The subunits are produced by endothelial cells (ECs) and megakaryocytes, where they combine to form multimers that range from 500,000 to 20,000,000 Daltons. VWF molecules are stored in alpha granules in platelets and Weibel-Palade bodies in ECs. ECs release ultralarge multimers of vWF into plasma, where they are normally degraded into smaller multimers by a VWF-cleaving protease, ADAMTS13 ( a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), in blood vessels with high shear stress. o VWF has both receptor for platelets’ GP 1b/V/IX complex during platelet adhesion. VWF also has receptor for GP IIb and IIIa of platelets , the reason why it also functions in platelet aggregation. o Levels of VWF vary by ABO blood type with group O individuals having lower levels of VWF that any other blood group type. o VWF is an acute-phase protein as is Factor VIII, and levels increase in pregnancy, trauma, infection, and stress. • FIBRINOGEN STRUCTURE AND FIBRIN FORMATION, FACTOR XIII • • • FACTOR XI AND THE CONTACT FACTORS • The contact factors are so named because they are activated by negatively charged foreign surfaces. Factor XIIa transforms Prekallikrein( a glycoprotein that circulates bound to HMWK, into its active form kallikrein, which cleaves HMWK to form bradykinin. Factor XII and prekallikrein are zymogens that are activated to become serine proteases: HMWK is a nonenzymatic cofactor. THROMBIN • The main enzyme in the coagulation mechanism. The primary function of thrombin is to cleave fibrinopeptides A (FPA) and B (FPB) from the alpha and beta chains of the fibrinogen molecule,triggering spontaneous fibrin polymerization and the formation of the fibrin clot. FPA and FPB are measurable in plasma and serves as a marker of thrombin activation. Thrombin amplifies the coagulation mechanism by activating cofactors V and VIII and factor XI by a positive feedback mechanism that serves to generate more thrombin. Thrombin also activates Factor XIII, which forms covalent bonds between the D domains of the fibrin polymer to cross-link and stabilize the fibrin clot. Thrombin initiates aggregation of platelets. Thrombin bound to thrombomodulin activates the protein C pathway to suppress coagulation and it activates the thrombinactivatable fibrinolysis inhibitor (TAFI) to suppress fibrinolysis. Elevated levels of TAFI is associated with an increased risk of venous thrombosis. Thrombin therefore plays a role in coagulation ( fibrin), platelet activation, regulation of coagulation activation ( protein C), and controlling fibrinolysis (TAFI). Because of its multiple autocatalytic functios, thrombin is considered the key protease of the coagulation pathway • Fibrinogen is the primary substrate of thrombin, which converts soluble fibrinogen to insoluble fibrin to produce a clot. Fibrinogen is also essential for platelet aggregation because in links activated platelets through their GP IIb/IIIa platelet fibrinogen receptor. Fibrinogen is a 340,000 Daltons glycoprotein synthesized in the liver. The normal plasma concentration of fibrinogen ranges from 200-400 mg/dL, the one with the highest concentration among the procoagulants in the blood.. It is an acute –phase reactant protein, whose level increases in inflammation, infection, and other stress conditions. Platelet alpha granules absorb, transport, and release abundant fibrinogen. The fibrinogen molecule is a mirror-image dimer, each half consisting of three non-identical polypeptides, designated Aa, Bb, and gamma, united by several disulphide bonds. The six N-terminals assemble to form a bulky central region called the E domain. The three carboxyl terminals on each outer end of the molecule assemble to form two D domains. Thrombin cleaves the FPA and FPB from the protruding N-terminals of each of the two alpha and beta chains of fibrinogen, reducing the overall molecular weight to 10,000 Daltons. The cleaved fibrinogen is called fibrin monomer. The exposed fibrin monomer alpha and beta chain ends (E domains) have an immediate affinity for portions of the D domain of neighboring monomers, spontaneously polymerizing to form fibrin polymer. Factor XIII is a heterodimer whose alpha subunit is produced mostly by megakaryocytes and monocytes, and whose beta subunit is produced in the liver. After activation by thrombin,factor XIIIa covalently cross-links fibrin polymers to form a stable insoluble fibrin clot. Factor XIIIa is a transgutaminase that catalyzes the formation of covalent bonds between the carboxyl terminals of gamma chains from adjacent D domains in the fibrin polymer. These bonds link the epsilon amino acid of lysine moieties and the gamma-amide group of glutamine units. Mutiple cross-links form to provide an insoluble meshwork of fibrin polymers linked by their D domains, providing physical strength to the fibrin clot. Factor XIIIa reacts with other plasma and other cellular structural proteins and is essential to wound healing and tissue integrity. • Cross-linking of fibrin polymers by factor XIIIa covalently incorporates fibronectin, plasma protein involved in cell adhesion, and alpha2- antiplasmin, rendering the fibrin mesh resistant to fibrinolysis. Plasminogen also becomes covalently bound via lysine moieties, as does TPA, a serine protease that ultimately hydrolyzes and activates bound plasminogen to activate fibrinolysis, thus breaking down the clot. • • COAGULATION SYSTEM • • The extrinsic, intrinsic pathways as well as the common pathways of coagulation can very well explain the clotting mechanism in vitro, so it is the prominent mechanism in clot-based assays. Formation of TF:VIIa has since proven to be the primary in vivo initiation mechanism for coagulation. Because TF is not present in blood, the TF pathway is called the extrinsic pathway. This pathway includes the factors VII, X, V, prothrombin and fibrinogen. HEMOSTATIC SYSTEM: CELL- BASED PHYSIOLOGIC COAGULATION • • • The Cascade theory, composed of the intrinsic, extrinsic, common pathways do not fully describe how coagulation occurs physiologically. These pathways are not distinct, independent, alternative mechanisms for generating thrombin but are actually interdependent. For example, a deficiency of factor VII in the extrinsic pathway can cause significant bleeding, even when the intrinsic pathway is intact.. Similarly, deficiencies of factors VIII and IX may cause severe bleeding regardless of the presence of a normal extrinsic pathway. In addition to the procoagulant and anticoagulant plasma proteins, normal physiologic coagulation requires the presence of two cell types for formation of coagulation complexes: cells that express TF ( usually extravascular) and platelets ( intravascular). Operationally, coagulation can be described as occurring in two phases: initiation, which occurs on tissue factor-expressing cells and produces 3% to 5% of the thrombin generated, and propagation, oc occurring on platelets, which produces 95% or more of the total thrombin. COAGULATION REGULATORY MECHANISM • • Inhibitors and their cofactors regulate serine proteases and cofactors in the coagulation system.They also provide feedback loops to maintain a complex and delicate balance between abnormal thrombosis and bleeding. These inhibitors, or natural anticoagulants, function to slow the activation of procoagulants and suppress thrombin production. They ensure that coagulation is localized and is not a systemic response, and they prevent excessive clotting or thrombosis. The principal regulators atr TFPI, antithrombin (AT), and activated protein C (APC). Acquired or inherited deficiencies of these proteins may be associated with increased incidence of venous thromboembolic disease because the hemostatic balance is shifted toward increased thrombin generation and coagulation rather than normal termination of the activated pathway. • • Tissue Factor Pathway inhibitor - a serine type Kunitztype serine protease inhibitor and is the principal regulator of the TF pathway. The Kunitz-2 domain binds to and inhibits factor Xa, and Kunitz-1 binds and inhibits the VIIa:TF complex. TFPI is synthesized primarily by ECs and also expressed on platelets. Protein S (PS) the cofactor of APC is also a cofactor of TFPI and enhances factor Xa inhibition byTFPI tenfold. Because of the inhibitory reaction of TFPI, the TF:VIIa:Xa reaction is short-lived. Once the TFPI shuts down the extrinsic tenase complex and Xa, additional Xa and IXa production shifts to the intrinsic pathway. Propagation of coagulation occurs as factor X is activated by IXa:VIII and more factorIX is activated by factor Xia Protein C Regulatory System - In intact blood vessels, where coagulation would be inappropriate, thrombin avidly binds the EC membrane protein thrombomodulin and triggers an essential coagulation regulatory system called the protein C system. The protein C system revises thrombin’s function from a procoagulant enzyme to an anticoagulant. Antithrombin III - first of the coaguylation regulatory proteins to be identified and the first to be assayed routinely in the clinical hemostasis laboratory. AT is a serine protease inhibitor( SERPIN) that binds and neutralizes serine proteases, including thrombin and factors IXa,Xa,Xia,XIIa, prekallikrein, and plasmin. AT requires heparin for effective anticoagulant activity. Heparin cofactorsII (HCII), an inhibitor that primarily targets thrombin,similarly requires heparin. AT activity is amplified 2000-fold by binding to heparin. Heparin induces a conformational change in the AT molecule that allows binding of activated coagulation factors, which cause3s them to be inactivated. The inhibitiuon of thrombin, factor X, and other serine proteases by AT is dependent on the length of the heparin chain; for thrombin, only the longer heparin chains are able to bind both molecules to produce inhibition of thrombin. When AT covalently binds thrombin, an inactive thrombinantithrombin complex (TAT) is formed, which is then released from the heparin molecule. Laboratory measurement of TAT is used as an indicator of thrombin generation. Other Serine protease Inhibitors (SERPIN) o Include ZPI, protein C inhibitor, alpha 1 protease inhibitor( alpha 1-antitrypsin),alpha2-macroglobulin, alpha2-antiplasminb, and PAI-1.