Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin.

We have developed an in vitro protocol for the measurement of clot-bound thrombin. This protocol uses a chromogenic substrate for thrombin and a microtiter plate reader and is suitable for screening inhibitors for thrombin that are directed to clot-bound thrombin. Clots were obtained after recalcification of human plasma. For the measurement of clot-bound thrombin, we read the optical density (OD) at 405 nm on a spectrophotometer and compared the results to that obtained with a standard curve of human alpha thrombin. We stopped the amidolytic reaction at 10 min because the optical density was linear until 20 min under our experimental conditions. We suggest that clot-bound thrombin can be measured using a chromogenic substrate specific for thrombin under our experimental condition.