A novel clotting assay for quantitation of plasma prothrombin (factor II) using Echis multisquamatus venom

Measuring plasma prothrombin activity seems useful for evaluating thrombotic risk and managing oral anticoagulant therapy as an adjunct to the international normalized ratio. Therefore, we designed a new plasma prothrombin assay based on the ability of Echis multisquamatus venom to activate prothrombin with only calcium as a cofactor. In this assay, 1 part of undiluted citrated plasma is added to 5 parts of a venom reagent and the clotting time is measured. The assay's advantages are that dilution of the test plasma is required only when prothrombin activity exceeds 100%, a single standard curve can be used over months for a given batch of stock reagent, and barium-adsorbed plasma is used for dilution of test plasma and construction of the standard curve, thus eliminating the need for prothrombin-deficient plasma. However, one should be aware of the following: (1) test samples must contain at least 200 mg/dL fibrinogen; and (2) when prothrombin concentrations were below 50%, the venom-based assay often gave values up to 10% higher than the thromboplastin-based assay. Values obtained in 262 plasma samples tested with the venom-based assay and with a thromboplastin-based prothrombin assay correlated well (r2 = 0.93).