Interaction between thrombin mutant W215A/E217A and direct thrombin inhibitor.

Thrombin mutant W215A/E217A acts as an anticoagulant when administered intravenously by activating plasma protein C in concert with endothelial thrombomodulin [1,2]. The double mutation in the catalytic domain of the W215A/E217A molecule leads to compromised interactions with fibrinogen and chromogenic thrombin substrates [1,3]. Among direct thrombin inhibitors (DTIs), argatroban exerts antithrombotic activity by a rapid binding to the catalytic domain of thrombin, while lepirudin and bivalirudin occupy both exosite I and catalytic domains [4]. We hereby report on the changes in DTI-interaction with W215A/E217A, using thrombin substrate hydrolysis [1,3], activated partial thromboplastin time (aPTT) and the Thrombinoscope methods (Thrombinoscope BV, Maastricht, Netherlands) [5]. Based on the inhibition of H-D-Phe-Pro-Arg-p-nitroanilide hydrolysis, argatroban and lepirudin interact with the catalytic domain of W215A/E217A with a 7000-fold reduced affinity relative to wild-type thrombin (Table 1) [6]. This reduction coincides with the 19 000-fold decrease in affinity for fibrinogen [1]. On the contrary, lack of inhibition of the substrate hydrolysis with bivalirudin suggests that W215A/E217A binds to bivalirudin utilizing exosite I without blocking the active site. This finding was confirmed in studies looking at the competitive inhibitory effect of bivalirudin on the activation of protein C by W215A/E217A in the presence of thrombomodulin (Table 1). Table 1 Binding of wild-type and mutant thrombin to thrombin inhibitors and substrates Although the direct interaction between W215A/E217A and DTIs seems to be weak, addition of W215A/E217A in DTI-treated plasma corrected prolongation of aPTT toward normal (Fig. 1). Prolonged aPTT with DTIs was not reversed with active site mutant S195A [7] or active-site blocked thrombin (wild-type thrombin inactivated with H-D-Phe-Pro-Arg-CH2Cl; FPR-WT). Inactive thrombin species, S195A and FPR-wild-type, occupy thrombin binding sites on fibrin(ogen) and cause dose-dependent inhibition of clotting [8]. It is thus speculated that the catalytic domain of W215A/E217A exerts some procoagulant function through activation of factor V [9]. Anticoagulant effects of DTIs have been shown to be mitigated in the presence of factor Va [10,11]. Fig. 1 Activated partial thromboplastin time, measured in normal volunteer plasma, spiked with a direct thrombin inhibitor (n = 8). Mean values (±SD) of activated partial thromboplastin time(aPTT) (in seconds). Note aPTT values were shortened by W215A/E217A . Using the Thrombinoscope system that continuously monitors thrombin generation based on the hydrolysis of fluorogenic substrate, Z-Gly-Gly-AMC (Bachem Bioscience, King of Prussia, Pennsylvania, USA), we showed that in plasma the combination of W215A/E217A (5µg/ml~135 nmol/l) and thrombomodulin (0.75µg/ml; Asahi Kasei Pharma, Oh-hito, Japan) [12], but not W215A/E217A alone exerted anticoagulant effects based on the reduced peak levels of thrombin generation (Fig. 2a). The addition of W215A/E217A was found to reverse prolonged thrombin generation lag time for all DTIs (Fig. 2). Reduced peak thrombin levels were not significantly recovered with W215A/E217A at therapeutic levels (0.5–1µg/ml) of argatroban (Fig. 2b) [13]. Our present data are in agreement with the reversal of melagatran-prolonged lag time in thrombin generation with factor Va [10]. For bivalent inhibitors, it is plausible that bivalirudin and lepirudin reduce the interaction of factor V with W215A/E217A by binding to exosite I [9]. FPR-wild-type (30µg/ml) reversed delayed onset of thrombin generation with bivalirudin, but clotting was extensively inhibited due to occupation of thrombin binding sites of fibrin with FPR-wild-type (Fig 1 and Fig 2c) [8]. Lepirudin obliterated thrombin generation, but this was restored toward normal after the addition of W215A/E217A, especially at therapeutic level (1µg/ml) of lepirudin (Fig. 2d). Fig. 2 Effects of W215A/E217A on thrombin generation. Panel a: a representative series of thrombin generation curves. The peak thrombin generation is slightly decreased by the addition of W215A/E217A, 5µg/ml, and modestly decreased by recombinant human . In summary, the catalytic-site double mutant, W215A/E217A, shortens DTI-prolonged aPTT and the onset of endogenous thrombin generation in vitro. Further modifications of the catalytic domain or exosites of thrombin molecule or both may lead to a development of antidotes for DTI-related bleeding complications [10].