Factor IXa variants resistant to plasma inhibitors enhance clot formation in vivo.

Factor IXa plays a pivotal role in coagulation by contributing to the activation of factor X via the intrinsic pathway. While antithrombin and other plasma inhibitors are thought to regulate factor IXa procoagulant function, the impact of factor IXa inhibition on thrombin generation and clot formation in vivo is unclear. Here, we generated factor IXa variants with altered reactivity to plasma inhibitors that target the factor IXa active site but maintain procoagulant function when bound to its cofactor, factor VIIIa. We found that select factor IXa variants (e.g., FIXa-V16L) have a prolonged activity half-life in plasma due, in part, to antithrombin resistance. Studies using hemophilia B mice show this delayed factor IXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of factor IX protein. Overall, the results demonstrate that regulation of factor IXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiologic regulation of factor IXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering inhibition of factor IXa could have therapeutic benefit.