Identification of the complementary binding domains of histidine-rich glycoprotein and factor XIIa responsible for contact pathway inhibition

Abstract

Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis.