Friday, November 22, 2024
11/22/2024
Clotting factor VIIa (FVIIa) initiates the activation of the coagulation cascade by binding to the procoagulant cofactor tissue factor. Recombinant FVIIa is a clinically approved drug for treating bleeding in hemophilia patients with inhibitors and other bleeding disorders. It is also used off-label to treat severe bleeding associated with surgery, liver disease, and intracerebral hemorrhage. We have discovered that FVIIa also binds to the anticoagulant cofactor, endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR modulates protein C/activated protein C-mediated anticoagulant pathway. Our studies also established that FVIIa-EPCR activates protease-activated receptor 1 (PAR1)-induced biased cell signaling, resulting in anti-inflammatory and vascular barrier protective effects. Interestingly, our recent studies revealed that FVIIa-EPCR-PAR1-mediated biased signaling induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). These vesicles are found to exhibit hemostatic and anti-inflammatory properties. EVs are increasingly recognized as important mediators of intercellular communication, play an important role in various pathophysiological processes, and likely have immense therapeutic potential. Understanding the biogenesis and release of FVIIa-generated EVs, characterizing their cargo, interactions with recipient cells, and their behavior in vivo is crucial for assessing their role in pathophysiology and fully capitalizing on their therapeutic and drug delivery potential. The proposed aims are designed to address these important knowledge gaps. Our overall hypothesis is that FVIIa-released EVs contribute to hemostatic, anti-inflammatory, and vascular barrier protective effects by communicating with other cell types by transferring their unique cargo. These EVs hold therapeutic potential in treating bleeding disorders, inflammation, and hemophilic arthropathy. The specific aims are, Aim 1: Investigate the hypothesis that FVIIa- released EEVs are unique and distinct from EEVs released by other coagulation proteases. Aim 2: Elucidate the mechanism of FVIIa-released EV biogenesis, phosphatidylserine (PS) enrichment of FVIIa-released EEVs, and their distribution and fate in vivo. Aim 3: Determine the role of FVIIa-released EEVs in hemostasis and inflammation and elucidate potential mechanisms. Aim 4: Determine the effect of FVIIa-released EEVs in the treatment of hemophilic arthropathy (HA) and explore the potential contribution of miR10a, found in the cargo of FVIIa-released EEVs, to this process. In these proposed studies, we will employ an unbiased omics approach to characterize FVIIa-released EEVs. We will also use loss- and gain-of-functional studies, unique transgenic mice, and murine model systems of bleeding, hemophilic arthropathy, and inflammation. The knowledge gained from our studies will not only lead to more efficient and cost-effective clinical treatments for hemophilia patients but also lead to novel therapeutic strategies for bleeding and inflammatory disorders. Our studies will contribute to a paradigm shift in our current understanding of proteases-induced cell signaling and their potential to affect cellular processes in distant cells through communication via EVs.
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