Understanding and Enhancing the Therapeutic Efficacy of Factor VIIa for Emergency Hemostasis

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Intracranial hemorrhage (ICH) is the second most common cause of stroke and results in the highest rates of stroke-related morbidity and mortality. While ICH can be spontaneous, it is also a well-known feature of severe traumatic brain injury (TBI). Each year 1.7 million Americans sustain a TBI and nearly half of these will result in long-term disability. In addition, TBI has become the signature wound of recent military operations and training with 17.3% of veterans meeting criteria for TBI during military service in Iraq and Afghanistan. Despite intensive research efforts and advances in critical care, the 30-day mortality rate from ICH has not changed significantly in the last 30 years. Current management remains largely restricted to optimizing cerebral perfusion pressure and providing supportive care. While the use of recombinant Factor VIIa (rFVIIa) as a hemostatic agent significantly reduces hematoma growth for patients with hemorrhagic stroke and TBI, the mortality benefit remains unclear. This is, in part, due to the significant risk of complications from diffuse inflammation and microvascular dysfunction that can exacerbate hemorrhage and cause secondary neuronal injury via mechanisms that are not amenable to surgical or hemostatic intervention. In addition, the use of rFVIIa is limited by a significant risk of thrombotic complications, particularly at higher doses. Therefore, a molecule with both hemostatic and anti-inflammatory activities could have distinct advantages over existing therapies by reducing both the primary and secondary complications of bleeding. The efficacy of rFVIIa is dependent on binding to activated platelets; however, previous attempts to improve this drug have failed, in part, because the mechanism of platelet-rFVIIa binding is not well understood. Therefore, the overall objective of the proposed studies is to elucidate the mechanisms of platelet-rFVIIa interaction as a means to design rFVIIa variants with enhanced hemostatic efficacy, reduced thrombotic risk, and potential anti-inflammatory and cytoprotective properties. Studies in the current proposal will focus on how procoagulant platelets store and regulate the surface expression of a novel protein that contributes to the binding and hemostatic activity of rFVIIa. These studies will use a combination of confocal microscopy and immunogold staining followed by transmission electron microscopy to characterize the subcellular localization of this protein and further define its origin, trafficking, and the potential regulation of its platelet-surface expression. This data will be enhanced by further studies that will utilize a chemical cross-linking and proteomics approach to identify additional partner(s) that make up the complex simultaneous interactions required for platelet-rFVIIa binding. Finally, this work will also determine the therapeutic potential of a novel Protein C-FVIIa chimera designed with the potential for increased hemostatic efficacy and reduced thrombotic risk relative to rFVIIa while retaining the anti- inflammatory and neuroprotective activities of Protein C. Flow cytometric analyses and enzyme activity assays will determine the platelet binding and activity of this chimera in vitro. Hemostatic efficacy and thrombogenic potential will be determined in vivo using murine saphenous vein bleeding and venous stasis models, respectively. The potential cytoprotective and anti-inflammatory effects will also be determined using cell culture and murine vascular permeability assays. When completed, this work will have a positive translational impact by elucidating the interactions necessary for platelet-rFVIIa binding and providing insights into how platelets regulate the surface expression of procoagulant proteins. In addition, the PC-FVIIa chimera not only represents a hemostatic agent that could be developed for clinical use, it also serves as a tool to address basic mechanisms of hemostasis and guide the design of other potential therapies to address the significant unmet need for better strategies to improve outcomes in veterans with ICH due to stroke or severe TBI.