The Role of Thrombin Exosites 1 and 2 in the Activation of Factor XI by Thrombin

Abstract

Factor XI (FXI) is the zymogen of the coagulation protease factor XIa (FXIa) that contributes to thrombin generation through the intrinsic pathway. FXI is activated by the contact pathway protease, factor XIIa (FXIIa), in a high molecular weight kininogen-dependent manner. It is also known to be activated by thrombin in a positive feedback reaction, however, the mechanism of this activation is not yet completely understood. Therefore, our objectives were to identify the role of polyanions in the thrombin mediated FXI activation, and the role of the thrombin exosites in the activation. To study this activation, we assessed the activation of FXI by thrombin in the presence and absence of the polyanions, dextran sulfate and polyphosphate (polyP). We utilized surface plasmon resonance to determine whether FXI and thrombin bind to the polyanions, and how the exosites effect thrombin’s ability to bind using thrombin exosite 1 and 2 variants. To investigate the role of the exosites in FXI activation, we analyzed the activation of FXI by the thrombin variants. In addition, we explored how inhibiting the thrombin exosites using DNA aptamers affects thrombin’s ability to bind to polyanions and activate FXI. We found that polyanions are required as a cofactor for the activation of FXI by thrombin, and stimulate the activation in a concentration dependent fashion, suggesting a template mechanism. Our findings also show that exosite 1 and 2 are required for thrombin to bind to polyanions, however, exosite 2 plays the predominant role in FXI activation. Our aptamer data showed that either exosite can be targeted to inhibit FXI activation. These findings enrich our understanding of the mechanism of FXI activation by thrombin and provides further insight on how to attenuate the activation for potential antithrombotic therapies.