PROJECT 2-PROJECT SUMMARY/ABSTRACT The coagulation system is tightly regulated to ensures hemostasis is properly achieved following injury. Factor V (FV) is a crucial component of this system playing both procoagulant and anticoagulant roles. Imbalanced expression and control of these activities can result in bleeding or thrombosis. Recent work by us and others suggests that the central B-domain of FV plays a key role in regulating its opposing functions. Activation of FV to FVa leads to the release of the B-domain, committing it fully to a procoagulant path resulting in rapid thrombin generation. However, FV also has anticoagulant properties, working with APC and TFPIα as a cofactor to inhibit coagulation. Our central hypothesis is that there are unique features of the FV B-domain that regulate the development of FV's procoagulant activity and contribute to function with APC and TFPIα. We propose that the B-domain acts as a gatekeeper for FV, controlling the balance between procoagulant and anticoagulant activity. There is limited information about the B-domain structure and little known about the mechanisms and structural elements that underlie these functions. This proposal aims to address these knowledge gaps and use the new information to improve clinical understanding and investigate potential therapies for bleeding or thrombosis. In Aim 1, we plan to determine the structure of the FV B-domain using atomic force microscopy. We will investigate how proteolysis affects its structure and examine how specific structural features regulate procoagulant function and interaction with TFPIα. We will test the hypothesis that the B-domain adopts a double lariat structure which is stabilized by certain sequences and disrupted by discrete proteolysis. The structural changes that occur in the B-domain as a result will be studied to understand interactions with FXa and TFPIα. In Aim 2, we propose to elucidate mechanisms and structural features of the anticoagulant functions of FV and its spliced variant FV-short, and to expand knowledge on their physiological effects. Using preliminary data, we will test the hypothesis that FV-short has a stronger anticoagulant effect when acting as a cofactor for APC compared to FV and investigate specific structural features that may enhance or inhibit this function. To achieve this, a series of FV and FV-short variants will be evaluated, and we will investigate procoagulant antibodies that bind to FV/FV-short and affect the APC and TFPIα anticoagulant pathways in plasma and in vivo. In Aim 3, we plan to advance work on new antibodies that target FV, FV-short or FVa to modulate its pro- or anticoagulant function. The goal is to use these antibodies to improve our understanding of these pathways and develop new therapeutic approaches. Our preliminary data support the hypothesis that FV-specific antibodies targeting its anticoagulant function may have potent procoagulant effects in vivo. By investigating the structure of ...