CA-1 method, a novel assay for quantification of normal prothrombin using a Ca2+ -dependent prothrombin activator, carinactivase-1.

We established a novel prothrombin assay, designated CA-1 method, for quantification of normal prothrombin in application of a Ca2+ -dependent prothrombin activator, carinactivase-1 (CA-1), found in the venom of Echis carinatus leucogaster. On microplate, thrombin converted from normal prothrombin in plasma sample by CA-1 cleaves a thrombin specific chromogenic substrate, t-butoxy-Val-Pro-Arg-p-nitroanilide and liberates p-nitroaniline. Then, the normal prothrombin level is decided by measuring the velocity of p-nitroaniline liberation. Normal prothrombin levels in plasma from warfarin-treated individuals were highly correlated with coagulant activities assayed by both prothrombin time and thrombotest. CA-1 method is not only a rapid and highly sensitive chromogenic microplate assay for quantification of normal prothrombin in the range of 10-200 ng/100 microl in plasma samples but also suitable for analyses of many samples in a short time. In addition, normal prothrombin levels obtained by CA-1 method are not inhibited by EDTA and heparin, which reduce prothrombin time and thrombotest activities. CA-1 method is a novel assay for monitoring coagulant activity in warfarin-treated individuals.