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Diagnostic Use of Venoms
The most common use of snake venom molecules as reagents in hemostasis is described below.
Fibrinogen Assessment
Fibrinogen promotes platelet aggregation and is converted to fibrin by thrombin, with formation of a hemostatic plug.
Reptilase Time
This is a modification of the thrombin time (TT) in which thrombin-like enzymes (serine proteases) replace thrombin. The most commonly used enzyme is Batroxobin, a 43 Kda protein from the venom of Bothrops atrox . It splits the Arg 16 –Gly 17 bond in the Aα-chain of fibrinogen with formation of fibrin monomer. Alternatively, Ancrod from Calloselasma rhodostoma is employed in the assay. Contrarily to thrombin, these enzymes cleave fibrinopeptide A, but not fibrinopeptide B, do not activate factors (F) V, FVIII, FXI, FXIII, and do not induce platelet aggregation. Importantly, they are not sensitive to heparin–antithrombin complex or hirudin. A second laboratory use for Batroxobin is to detect antithrombin activity. Using these enzymes, plasma can be prepared free of fibrinogen without addition of thrombin, which would otherwise react with antithrombin and interfere with the assay.
Principle of the Tests and Interpretation
Reptilase reagent is added to platelet poor plasma and clotting time (CT) is measured; phospholipid or Ca 2+ is not required. The RT is usually performed together with TT, and results from both tests are complementary in the interpretation of a coagulation disorder. A prolonged RT and TT are seen in inherited and acquired dysfibrinogenemia. Both tests may be prolonged following thrombolytic therapy or in disseminated intravascular coagulation, due to the high levels of fibrin-degradation products, which inhibit the fibrin polymerization step, or due to hypofibrinogenemia. Other conditions that might be associated with prolonged RP and TT are hypoalbuminemia seen in nephrotic syndrome, in liver disease due to dysfibrinogenemia, and in multiple myeloma due to paraproteins affecting fibrinogen polymerization. The RT is not affected by low-molecular-weight heparin or unfractionated heparin, direct thrombin inhibitors, and warfarin, in contrast to the TT.
Meizothrombin Assessment
Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of FXa and FVa and phospholipid (prothrombinase complex).
Ecarin Clotting Time
Several prothrombin activators have been identified in snake venoms and classified in four different types, according to structure, function, and cofactor requirements. Ecarin is a 55 kDa metalloprotease from the venom of Echis carinatus and specific activator of prothrombin. It activates prothrombin independently of Ca 2+ , phospholipids, and FV (do not require a cofactor). Ecarin cleaves prothrombin at Arg 323 –Ile 324 producing meizothrombin, an intermediate which is finally converted into α-thrombin by autolysis. Thrombin is detectable in a clotting assay known as Ecarin clotting time (ECT) or with chromogenic substrates in an assay known as Ecarin chromogenic assay (ECA). Importantly, meizothrombin binds to and is inhibited by hirudin but is unaffected by heparin/antithrombin because of steric hindrance. Therefore, the ECT is a test to determine meizothrombin production and can be used to detect direct thrombin inhibitors (e.g., hirudin) in citrated and heparinized blood. ECT is also employed to determine lupus anticoagulant (LA).
Principles of the Test and Interpretation
Ecarin is added to plasma and ECT determined turbidimetrically. In the presence of hirudin, meizothrombin inhibition leads to prolongation of clot formation for hirudin concentrations of 0.05–5.0 μg/mL. However, plasma levels >2.5 μg/mL found in cardiac surgery require a modification of ECT in which the blood sample is diluted 1:1. ECT is particularly useful in clinical practice because the activated partial thromboplastin time (aPTT) shows a linear dose-response curve for hirudin concentrations up to 1 μg/mL, whereas TT and activated clotting time show a nonlinear correlation. The ECT is relatively insensitive to variations in clotting factors, including fibrinogen and prothrombin. In the ECA, generation of meizothrombin is measured using a specific chromogenic substrate.
Factor V Assessment
FVa is a cofactor for the prothrombinase complex. RVV-V is a 27 kDa serine proteinase from the Russell’s viper venom Daboia russelli snake, which converts FV to FVa. RVV-V may be used to determine FV levels but is most commonly used in the determination of LA and resistance to activated protein C (APCR) (see below).
Factor X Assessment
FXa activates prothrombin in the presence FVa, phospholipid, and Ca 2+ (prothrombinase complex).
RVV-X From Russell’s Viper Venom
RVV-X is a 120 kDa metalloprotease from the D. russelli snake and consists of two peptide chains with a molecular weight of 60 kDa each. It activates factor X and is strictly dependent on Ca 2+ , FV, prothrombin, and phospholipids. RVV-X is used to quantitatively convert FX to FXa, which can be determined by clotting-based assay or with chromogenic substrates. RVV-X activator is also used to test for LA.
Principles and Interpretation of the Test
The principle of the RVV-X is similar to that of prothrombin time (PT)–based FX determination. An aliquot of the FX-deficient substrate plasma is mixed with an aliquot of the control or test plasma, and RVV-X and phospholipid substitute are added. Clotting is initiated by the addition of Ca 2+ and the time to clot formation is recorded. On a log–log graph, CT for the PT is plotted against each dilution and the results compared with a log–log graph plotted with FX standard and results interpolated. Low FX levels may be observed in patients with vitamin K deficiency, FX deficiency, acquired factor inhibitors, and amyloidosis due to absorption of FX in amyloid fibrils. The chromogenic assay utilizes a specific substrate, which is cleaved by FXa generated by RVV-X, producing color that is detected spectrophotometrically.
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