Coagulation factor Vlla finds increasing therapeutic application in patients with severe bleeding disorders. We have recently shown that Vlla participates in a unique signaling mechanism of the upstream coagulation reaction in which Xa, in the transient TF-Vlla-Xa complex, activates protease activated receptor (PAR) 1 or 2. The importance of these potentially proinflammatory signaling events for the safety of Vlla therapy is unexplored. This application proposes to characterize the molecular details of upstream coagulation protease signaling and to develop an in vivo model that is suitable to assess the role of Vlla in proinflammatory cell signaling.
Aim 1 is to characterize the structural determinants in Vlla that stabilize the signaling competent TF-Vlla-Xa complex. We hypothesize that certain residue side chains have distinct roles in supporting signaling of the TF-Vlla-Xa complex versus promoting the generation of Xa, which upon release from the complex triggers the downstream coagulation effector cascade. The goal is to identify and characterize Vlla mutants with selective elimination of either procoagulant or signaling activity.
Aim 2 is to define the structural basis of PAR specificity of upstream coagulation proteases. We hypothesize that the selective activation of PAR1 by Xa, but not Vlla, results from an unconventional docking of the hirudin-like sequence of PAR1 to basic residues that are unique to Xa. By mutagenesis we will define the structural features that support the Xa-mediated activation of PAR1. Mutants of Xa are further characterized in a primary cell endothelial model in which we provided evidence that Xa is a relevant activator of PAR1.
Aim 3 is to establish a mouse model to characterize signaling of Vlla in vivo. We propose to exploit the unique biochemical properties of Vlla to administer mutants with altered signaling or procoagulant properties to a mouse model of inflammatory PAR signaling. This approach promises to rapidly bridge the biochemical studies to a validation of the mechanistic principles in vivo, providing fundamental insight into the structural basis and physiological relevance of upstream coagulation protease signaling.
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