Inhibition of Thrombin and Its Generation By Recombinant Thrombomodulin and Activated Protein C in Whole Blood and Plasma Based Systems

Abstract

Introduction:Recombinant thrombomodulin (rTM) and activated protein C (APC) are important modulators of coagulation and fibrinolysis. Both agents have been developed for various clinical indications. Beside their direct effect on coagulation, these agents also modulate other components of hemostatic processes to mediate their pharmacologic actions such as the direct inhibition of proteases and their generation.Aim:The aim of this study is to compare the differential inhibition of thrombin and its generation by rTM (ART-123, Asahi Kasei, Tokyo, Japan) and a plasma derived APC (Hematologic Technologies, Burlington, VT, USA).Methods:The inhibitory effects of APC and rTM on thrombin were measured in a plasma based and fluorogenic substrate assays. In the whole blood studies, native blood from healthy volunteers (n=10) was supplemented with rTM and APC at concentrations levels of 0-1.0 ug/ml. Thrombin generation was triggered by adding diluted APTT activator (Triniclot) and the reaction was stopped after 3 minutes. Thrombin generation markers such as F1.2 and TAT were measured using commercially available ELISA methods. In the citrated plasma studies both agents were supplemented at 0-1.0 ug/ml. Thrombin generation studies were carried out using a flouremtric assay (TGA, Techniclone, Vienna, Austria). Lag time peak thrombin generation and area under the curve (AUC) were measured. The IC50 values for each of the agents was calculated.Results:APC did not have any direct inhibitory effects on thrombin in both the clotting and amidolytic assays. However, rTM produced a concentration dependent inhibition of thrombin in both the clot based and amidolytic assays. In the whole blood systems both the APC and rTM produced a concentration dependent inhibition of thrombin generation markers such as F1.2 and TAT. APC produced a slightly stronger inhibition of stronger inhibition of TG as measured by F1.2, IC50 = 0.55 ug/ml compared with thrombomodulin, IC50 = 0.70 ug/ml. In the plasma based system the IC50 for peak thrombin inhibition was 0.46 ug/ml for APC and 0.97 ug/ml for rTM. Additional differences in the lag phase and AUC profiles were also noted. Conclusions: Beside the direct anticoagulant effects both the APC and rTM are capable of inhibiting the TF mediated thrombin generated in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in TG assays in both plasma and whole blood. .Conclusions:In contrast to APC, rTM produces direct inhibitory effects on thrombin in both the amidolytic and clot based assays. Both APC and rTM are capable of inhibiting the TF mediated thrombin generation in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in thrombin generation assays in both plasma and whole blood. These studies suggest that modulation of thrombin and other proteases contribute to the clinical effects of both rTM and APC. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.