Effect of sera on thrombin activation rate constants: a one-stage assay for the extrinsic system

A one-stage chromogenic assay sensitive to all factors of the extrinsic system has been developed. Diluted plasma is combined with tissue thromboplastin in the presence of S-2238, a thrombin-sensitive substrate. After a lag phase, log (A405/min) is linear with time up to the maximal thrombin concentration. The linear slope, b, is called the thrombin activation rate constant (TARC). Log b, or b, is linearly related to log transformations of plasma dilutions, of factor concentrations, of dicumarolized plasmas, and of one-stage prothrombin times. Since the lag phase can vary from 2 to more than 10 minutes, it is difficult to perform the assay on current automated equipment. Results show that factors VII and X affect the lag phase, while factors V and II do not. Small amounts of sera or thrombin, to a lesser extent, can shorten the lag phase to near zero without altering the relationships between TARC and plasma dilutions and between TARC and prothrombin times for dicumarolized plasmas and for dilutions of factor-deficient plasmas. The only effects of some sera are to increase b by approximately 20 percent. Successful sera can be made from clotted whole blood or supernatants of sera from citrated plasma clotted with tissue of partial thromboplastins. These sera appear to have minimal amounts of factor X, undetectable prothrombin, and undetectable free thrombin. The sera contain excesses of factor VII and/or factor VIIa and nearly 20 percent of factor V. If the sera activity arises from the extrinsic system, it is probably due to factor VIIa.