Secondary Hemostasis 2 - Introduction PDF

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CENTRAL LUZON DOCTORS’ HOSPITAL EDUCATIONAL INSTITUTION Romulo Hi-way, San Pablo, Tarlac City, Tarlac, Philippines MEDICAL TECHNOLOGY DEPARTMENT

MODULE IN HEMATOLOGY 2 --SECONDARY HEMOSTASIS-(PART TWO)

Prepared by: Alton Joshua S. Masanque, RMT, DTA

FIBRINOLYSIS Fibrin clots are temporary structures that seal off a damaged area until healing can take place. Fibrinolysis is the physiological process that removes insoluble fibrin deposits by enzymatic digestion of the stabilized fibrin polymers. As healing occurs, the clots themselves are dissolved by plasmin. Plasmin digests fibrin and fibrinogen by hydrolysis to produce progressively smaller fragments. This slow-acting process gradually dissolves away the clot as tissue repair is taking place, with the particulate matter being phagocytized by the mononuclear phagocytic system. Two activators of fibrinolysis, TPA and UPA, are released in response to inflammation and coagulation. Inactive plasminogen circulates in the plasma until an injury occurs. The activators of plasminogen consist of endogenous and exogenous groups. Plasminogen activation to plasmin is the result of the activity of a number of proteolytic enzymes. These enzymes, the kinases, are referred to as the plasminogen activators. Plasminogen activators are found in various sites, such as the vascular endothelium or lysosomal granules, and biological fluids. At least two forms of tissue activators have been described: those that seem related to urokinase, a urinary activator of plasminogen, and those unrelated to urokinase. The activators unrelated to urokinase include thrombin, bacterial products such as streptokinase from beta-hemolytic streptococci, and staphylokinase. Plasma activators of plasminogen include plasma kallikrein, activated plasma thromboplastin antecedents (factor XI), and activated Hageman factor (factor XIIa). Small amounts of plasmin become trapped in the clot. The specificity of plasmin ensures that clot dissolution occurs without widespread proteolysis of other proteins. Plasmin also activates the complement system, liberates kinins from kininogen, and can hydrolyze coagulation factors V, VIII, and XII. Further clot formation is impeded by antiplasmins and naturally occurring inhibitors, some of which prevent the activation of plasminogen. The naturally occurring inhibitors include AT-III, alpha-2 macroglobulin inhibitor, and alpha-1 antitrypsin. Plasmin is not normally found in plasma because it is neutralized by an excess of inhibitors.

PLASMINOGEN AND PLASMIN Plasminogen is a 92,000 Dalton plasma zymogen produced by the liver. It is a singlechain protein possessing five glycosylated loops termed kringles. Kringles enable plasminogen, along with activators TPA and UPA, to bind fibrin lysine molecules during polymerization. This fibrin-binding step is essential to fibrinolysis. Fibrin-bound plasminogen becomes converted into a two-chain active plasmin molecule when cleaved between arginine at position 561 and valine at position 562 by neighboring fibrin-bound TPA or UPA. Plasmin is a serine protease that systematically digests fibrin polymer by the hydrolysis of arginine-related and lysine-related peptide bonds. Bound plasmin digests clots and restores blood vessel patency. Its localization to fibrin through lysine binding prevents systemic activity. As fibrin becomes digested, the exposed carboxy-terminal lysine residues bind additional plasminogen and TPA, which further

accelerates clot digestion. Free plasmin is capable of digesting plasma fibrinogen, factor V, factor VIII, and fibronectin, causing a potentially fatal primary fibrinolysis. However, plasma a2antiplasmin rapidly binds and inactivates any free plasmin in the circulation.

OTHER SYSTEMS AND INHIBITORS KININ SYSTEM This system is activated by both the coagulation and fibrinolytic systems. Fletcher factor (prekallikrein) and Fitzgerald factor (HMWK) are also needed to enhance or amplify the contact factors involved in the intrinsic system. Factor XIIa in the presence of HMWK converts prekallikrein to kallikrein. Kallikrein feeds back to accelerate the conversion of factor XII to XIIa, which accelerates the intrinsic system processes. Activation of factor XII acts as the mutual path between many components of the hemostatic mechanism, including the fibrinolytic system, the kinin system, and the complement system.

COMPLEMENT SYSTEM Complement facilitates cell membrane lysis of antibody-coated target cells. Two independent pathways of complement activation, the classic and alternate pathways, can occur along with a common cytolytic pathway. Plasmin activates complement by cleaving C3 into C3a and C3b. C1 esterase inhibitor inactivates complement and also has a role in hemostasis. PROTEASE INHIBITORS Because the fibrinolytic system is activated when the coagulation cascade is activated, extra fibrin is degraded and eliminated along with some coagulation factors. Enzymes such as plasmin and kallikrein still circulate until they are eliminated by various mechanisms: liver hepatocytes, mononuclear phagocytic cells, or serine protease inhibitors present in the plasma. Serine protease inhibitors attach to various enzymes and inactivate them. Serine protease inhibitors includes: PROTEIN C REGULATORY SYSTEM During coagulation, thrombin propagates the clot as it cleaves fibrinogen and activates factors V, VIII, XI, and XIII. In intact normal 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 anticoagulant system. The protein C system revises thrombin’s function from a procoagulant enzyme to an anticoagulant. EC protein C receptor (EPCR) is a transmembrane protein that binds both protein C and APC adjacent to the thrombomodulin-thrombin complex and augments the action of thrombin-thrombomodulin at least fivefold in activating protein C to a

serine protease APC dissociates from EPCR and binds its cofactor, free plasma protein S. The stabilized APC-protein S complex hydrolyzes and inactivates factors Va and VIIIa, slowing or blocking thrombin generation/coagulation. PROTEIN S REGULATORY SYSTEM Protein S, the cofactor that binds and stabilizes APC, is synthesized in the liver and circulates in the plasma in two forms. About 40% of protein S is free, but 60% is covalently bound to the complement control protein C4b-binding protein (C4bBP). Bound protein S cannot participate in the protein C anticoagulant pathway; only free plasma protein S can serve as the APC cofactor. Protein S-C4bBP binding is of particular interest in inflammatory conditions because C4bBP is an acute phase reactant. When the plasma C4bBP level increases, additional protein S is bound, and free protein S levels become proportionally decreased, which may increase the risk of thrombosis. Chronic acquired or inherited protein C or protein S deficiency or mutations of protein C, protein S, or factor V compromise the normal downregulation of factors Va and VIIIa and may be associated with recurrent venous thromboembolic disease. a2-ANTIPLASMIN a2-Antiplasmin (AP) is synthesized in the liver and is the primary inhibitor of free plasmin. AP is a serine protease inhibitor with the unique characteristic of both N- and C-terminal extensions. During thrombus formation, the N-terminus of AP is covalently linked to fibrin by factor XIIIa. The Cterminal contains lysine, which is capable of reacting with the lysine-binding kringles of plasmin. Free plasmin produced by activation of plasminogen can bind either to fibrin, where it is protected from AP because its lysine-binding site is occupied, or to the C-terminus of AP, which rapidly and irreversibly inactivates it. Thus, AP with its C-terminal lysine slows fibrinolysis by competing with lysine residues in fibrin for plasminogen binding and by binding directly to plasmin and inactivating it. The therapeutic lysine analogues, tranexamic acid and eaminocaproic acid, are similarly antifibrinolytic through their affinity for kringles in plasminogen and TPA. Both inhibit the proteolytic activity of plasmin.

FIBRIN DEGRADATION PRODUCTS Plasmin cleaves fibrin and produces a series of identifiable fibrin fragments: X, Y, D, E, and D-D. Several of these fragments inhibit hemostasis and contribute to hemorrhage by preventing platelet activation and by hindering fibrin polymerization. Fragment X is described as the central E domain with the two D domains (D-E-D), minus some peptides cleaved by plasmin. Fragment Y is the E domain after cleavage of one D domain (D-E). Eventually these fragments are further digested to individual D and E domains. The D-D fragment, called D-dimer, is composed of two D domains from separate fibrin molecules cross-linked by the action of factor

XIIIa. Fragments X, Y, D, and E are produced by digestion of either fibrin or fibrinogen by plasmin, but D-dimer is a specific product of digestion of cross-linked fibrin only and is therefore a marker of thrombosis and fibrinolysis— that is, thrombin, factor XIIIa, and plasmin activation. The various fragments may be detected by quantitative or semiquantitative immunoassay to reveal fibrinolytic activity. D-dimer is separately detectable by monoclonal antibody for D-dimer antigen, using a wide variety of automated quantitative laboratory immunoassays and other formats including point-of-care tests performed on whole blood. The Ddimer immunoassay is used to identify chronic and acute DIC and to rule out venous thromboembolism in suspected cases of deep venous thrombosis or pulmonary embolism.

LABORATORY TESTS FOR BLOOD COAGULATION FACTORS ACTIVATED PARTIAL THROMBOPLASTIN TIME The aPTT procedure measures the time required to generate thrombin and fibrin polymers via the intrinsic and common pathways. In the aPTT assay, calcium ions and phospholipids that substitute for platelet phospholipids are added to blood plasma. In vitro, the activation of factor XII to XIIa, prekallikrein to kallikrein, and factor XI to XIa occurs on the negatively charged glass surface. The generation of fibrin is the end point. The aPTT assay reflects the activity of prekallikrein, HMWK, and factors XII, XI, IX, VIII, X, V, II, and I. aPTT may be prolonged because of a factor decrease, such as fibrinogen (factor I), or the presence of circulating anticoagulants. The reference range for aPTT is less than 35 seconds (depending on the activator used). PROTHROMBIN TIME The PT procedure evaluates the generation of thrombin and the formation of fibrin via the extrinsic and common pathway. Thromboplastin reagent is used for this assay. Thromboplastin can be prepared by various methods: tissue extraction of rabbit brain or lung, tissue culture, and molecular methods. Thromboplastin reagent is a mixture of tissue factor, phospholipid, and calcium ions and is used to initiate clotting measure as the PT. Thromboplastin forms complexes with and activates factor VII. This provides surfaces for the attachment and activation of factors X, V, and II. Thromboplastin, derived from tissues that supply phospholipoprotein, and calcium are added to the blood plasma. The time required for the fibrin clot to form is measured. Reference ranges are from 10 to 13 seconds. Prolonged results can indicate a deficiency of one or more factors in the extrinsic pathway: factors VII, X, V, and II or I. Prolonged values will be seen if an oral anticoagulant such as coumarin or a coumarincontaining substance (e.g., rat poison) is ingested. Carriers of a point mutation in the prothrombin gene, Prothrombin 20210 discovered in 1996, demonstration increased prothrombin activity. There is no screening

test for this mutation, but it can be investigated using molecular techniques if clinical signs and symptoms are suggestive of a defect. THROMBIN TIME The thrombin time test determines the rate of thrombininduced cleavage of fibrinogen to fibrin monomers and the subsequent polymerization of hydrogen-bonded fibrin polymers to form an insoluble fibrin clot. The normal value is less than 20 seconds. Prolonged results will be seen if the fibrinogen concentration is less than 100 mg/dL. Abnormal results will also be encountered in the presence of thrombin inhibitors or substances that interfere with fibrin formation (e.g., heparin, fibrin degradation products), or high concentrations of immunoglobulins that interfere with fibrin monomer polymerization such as in cases of multiple myeloma. ANTI-Xa The Chromogenic anti-Xa method for monitoring low– molecular-weight heparin and unfractionated heparin is another assay. This automated assay can replace the aPTT for monitoring unfractionated heparin because it eliminates the variability seen with aPTT results. MIXING STUDY A mixing study can be used in the case of a prolonged aPTT. The principle of the mixing study relies on a 1:1 mix of a normal patient plasma added to the patient’s test plasma. The aPTT is repeated, and if the results are normal, it suggests a coagulation factor deficiency. If there is no correction of the patient’s aPTT, the presence of an inhibitor is suggested.

ANTICOAGULANTS TRADITIONAL ANTICOAGULANTS WARFARIN The traditional oral anticoagulant is warfarin (Coumadin). Warfarin drugs are vitamin K antagonists that interfere with the normal synthesis of factors II, VII, IX, and X as well as proteins C and S. These drugs cause incomplete coagulation because they lack calcium-binding sites and cannot form enzyme substrate complexes. Thus, these factors are unable to function as procoagulants or anticoagulants. Biological activity is significantly decreased, as revealed by the PT. The onset of action of most warfarin derivatives is between 8 and 12 hours. The maximum effect occurs in approximately 36 hours, and the duration of action is approximately 72 hours. The PT, used to adjust the dose of oral anticoagulants, should be reported according to the INR, not the PT ratio or the PT expressed in seconds. The INR is essentially a corrected PT that adjusts for the several dozen

assays used in North America and Europe. Oral anticoagulant therapy monitoring in patients with lupus inhibitors has presented problems for some laboratories. The PT can be prolonged in patients with antiphospholipid antibody syndrome for a variety of reasons: Antibodies produced in this syndrome are directed toward phospholipidbinding proteins including prothrombin. LA or inhibitor interferes with the phospholipid in the in vitro assays of PT and aPTT. HEPARIN Heparin anticoagulation is the mainstay of immediate therapy for acute PE. Heparin has no anticoagulant activity of its own but acts as an anticoagulant by accelerating the binding of antithrombin to target enzymes (e.g., thrombin and factor Xa). Heparin is termed an antithrombin because it helps to prevent new thrombus formation and buys time for endogenous fibrinolytic mechanisms to lyse the clot. Heparin can cause bleeding, thrombocytopenia, and osteopenia. Before initiating heparin, patients should be screened for clinical evidence of active bleeding. The baseline laboratory evaluation should include complete blood count (CBC), platelets, PTT, PT, stool analysis for occult blood, and urine dipstick for hematuria. Heparin anticoagulation is used during percutaneous transluminal coronary angioplasty (PTCA) and cardiopulmonary bypass (CPB) to prevent clot formation. The activated clotting time (ACT) has been used for more than 25 years to assess the degree of anticoagulation in heparinized patients. Typically, ACTs are monitored to establish a minimum target ACT to ensure adequate anticoagulation. The ACT was one of the first coagulation tests to be offered at the point care, for example, operating room, cardiac catherization, or hemodialysis, and other interventional procedures that require large doses of heparin. Typically, the ACT is measured prior to and immediately after heparinization. Subsequent testing is performed to ensure that adequate anticoagulation continues or additional heparin is administered. OTHER ANTITHROMBIN-DEPENDENT INHIBITORS DANAPAROID (ORGARAN) Danaparoid is a mixture of heparinoids which only accelerates the binding of factor Xa to antithrombin and possesses no anticoagulant activity of its own. It is monitored by chromogenic assay.

FONDAPARINUX (ARIXTRA) Fondaparinux is a synthetic pentasaccharide that accelerates the binding of antithrombin to activated factor Xa. It has no antithrombin activity. Although this drug usually does not require monitoring, it is recommended that it be assayed by a system based on the inhibition of factor Xa, if monitoring is needed.

DISORDERS OF THE SECONDARY HEMOSTASIS Bleeding and defective fibrin clot formation are frequently related to a coagulation factor. Disorders of the blood coagulation factors can be grouped into three categories: Defective production Excessive destruction or consumption Pathological inhibition DEFECTIVE PRODUCTION VITAMIN K DEFICIENCY A condition of defective production may be related to a deficiency of vitamin K. The synthesis of vitamin K and dependent factors can be disrupted because of disease or drug therapy (e.g., cephalosporin antibiotics). Vitamin K deficiencies are also encountered in neonates, malabsorption syndrome, biliary obstruction, and patients taking oral anticoagulants. Vitamin K depletion develops within 2 weeks if both intake and endogenous production are eliminated. Factors II, VII, IX, and X are vitamin K dependent. Factor VII has the shortest half-life and usually declines in the early stages of vitamin K depletion. A mild deficiency of vitamin K may present as an asymptomatic prolongation of a patient’s PT assay. SEVERE LIVER DISEASE Because the liver is the primary site of synthesis of coagulation factor, severe liver disease can cause defective production of coagulation factors. Severe liver disease may produce decreased plasma levels of fibrinogen, although low levels of fibrinogen rarely produce hemorrhage. In patients with liver disease, the PT is noticeably prolonged, whereas the aPTTs are variable. HEREDITARY CLOTTING DEFECTS Classic hemophilia (hemophilia A) and von Willebrand disease are examples of hereditary disorders that represent functionally inactive factor VIII.

HEMOPHILIA Hemophilia has been used as a paradigm for understanding the molecular pathological processes that underlie hereditary disease. The cloning of factor VIII facilitated the identification of mutations that lead to hemophilia A, an inherited deficiency of factor VIII coagulant activity that causes severe hemorrhage. Two types of mutations dominate the defects identified so far: gene deletions and point mutations. Gene deletions are associated with severe hemophilia A in which no factor VIII circulates in the blood. To date, approximately 50 deletion mutations in the gene for factor VIII have been characterized at the molecular level, and 34 independent deletion mutations in the factor IX gene have been found to be the cause of hemophilia B. Point mutations, in which a single base in DNA is mutated to another base, represent a second type of mutation that causes hemophilia. Individuals with hereditary clotting defects may be either genetically homozygous or heterozygous carriers of the trait. The level of factor activity ranges from 0% to 25% in persons homozygous for the trait and from 15% to 100% in persons heterozygous for the trait. Defects of this origin may result from the decreased production of a clotting factor, factor VIII, or the production of functionally inactive molecules of the clotting factor. Hemophilia A, a sex-linked homozygous disorder expressed in males, occurs in 1 in 10,000 males. VON WILLEBRAND DISEASE In 1926, Erik von Willebrand first described a hemorrhagic disorder characterized by a prolonged bleeding time and an autosomal inheritance pattern that distinguished the disease from classic hemophilias. In the early 1950s, an additional component of the disease was identified: a deficiency of factor VIII procoagulant activity. These and other observations distinguish von Willebrand disease from classic factor VIII:C deficiency (hemophilia A). In addition...