The factor IXa heparin-binding exosite is a novel antithrombotic target

Heparin is a heterogeneous mixture of oligosaccharide chains with extensive clinical uses. The antithrombotic activity of heparin is dependent on multiple mechanisms of action, including binding to an exosite on factor IXa that overlaps with the cofactor (factor VIIIa) interactive site, disrupting the critical protein-protein interaction between factor IXa and factor VIIIa A2 domain. This exosite represents the molecular target for antithrombin-independent inhibition of the intrinsic tenase complex. Modeling of coagulation cascade indicates that factor X activation by intrinsic tenase complex is the rate-limiting step for thrombin generation. We hypothesized that the factor IXa heparin-binding exosite is a critical regulator of coagulation and represents a novel antithrombotic target. The role of the heparin-binding exosite in thrombin generation was assessed by fluorogenic assay and Western blot analysis with recombinant factor IX(a) possessing mutations that enhance or reduce stability of the protease-factor VIIIa A2 domain interaction. The dramatic effect of these mutations on thrombin generation emphasizes the critical role of the factor IXa heparin-binding exosite in physiologic regulation of coagulation. Depolymerized holothurian glycosaminoglycan (DHG) possesses antithrombin-independent antithrombotic properties and inhibits the intrinsic tenase complex by targeting the factor IXa heparin-binding exosite. Recombinant factor IX(a) with reduced heparin affinity demonstrated resistance to DHG inhibition of thrombin generation. Thus the factor IXa heparin-binding exosite is the molecular target for DHG inhibition of thrombin generation. The effect of unfractionated heparin (UFH), low-molecular-weight-heparin (LMWH), super-sulfated LMWH, and Fondaparinux on inhibition of plasma thrombin generation was similarly evaluated. In the absence of antithrombin, LMWH effectively inhibits thrombin generation within the therapeutic range, suggesting the potential therapeutic relevance of antithrombin-independent effects. Recombinant factor IX with reduced heparin affinity demonstrates resistance to inhibition of thrombin generation by LMWH and super-sulfated LMWH, confirming the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition by heparin. Thus, the factor IXa heparin-binding exosite is the molecular target for glycosaminoglycan-mediated, serpin-independent inhibition of thrombin generation, a mechanism that has both physiologic and therapeutic applications. Targeting this exosite with small molecules or synthetic glycosaminoglycans will lead to discovery of novel antithrombotic reagents with a wider therapeutic window and reduced bleeding risk.