Antithrombin-resistant prothrombin Yukuhashi mutation also causes thrombomodulin resistance in fibrinogen clotting but not in protein C activation

Abstract

IntroductionProthrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)–protein C (PC) anticoagulant system have not been previously elucidated. Materials and MethodsWe prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM. Result and ConclusionsrTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.