Mitochondrial fission proteins shape platelet production and function

Abstract Mitochondrial dysfunction is associated with numerous diseases, including disorders of thrombosis and hemostasis. While platelets inherit fully functional mitochondria from megakaryocytes, the mechanisms for preserving mitochondrial integrity during megakaryocyte development and their loading into platelets are not clear. Genetic studies have linked the mitochondrial fission protein Dynamin Related Protein 1 (DRP1) with platelet counts, size, and functional responses in diverse populations. Our preliminary data show that loss of DRP1 in human and mouse megakaryocytes profoundly reshapes mitochondrial morphology in megakaryocytes, culminating in platelets loaded with excessive and massive mitochondria. Platelet size was significantly increased, and platelet counts decreased, mirroring human association studies. Moreover, platelet functional responses were also compromised, pointing to a role for mitochondrial fission in multiple aspects of megakaryocyte and platelet biology. In Aim 1 we propose that basal DRP1-mediated fission maintains mitochondrial integrity during megakaryocyte development, while heightened fission at proplatelet production reshapes mitochondria for loading into proplatelets and subsequent platelet release. Aim 2 seeks to elucidate how loss of DRP1 and other fission proteins alters platelet function, hemostasis, and thrombosis through mitochondrial and non- mitochondrial mechanisms. These aims integrate mouse models and human studies, including platelet/megakaryocyte specific DRP1/FIS1 knockouts, newly developed DRP1 inhibitors, and CRISPR edited primary CD34+ cell derived megakaryocytes. Aim 3 employs a novel high-throughput CRISPR functional assay to comprehensively evaluate the impact of all known genes in the mitochondrial fusion/fission pathways on platelet-like functional responses in megakaryocytes. This research is pivotal and innovative: we will examine mitochondrial fission, a novel pathway regulating loading of mitochondria into platelets to modulate platelet size and function. Our studies will shed light on why human DRP1 variants are associated with platelets phenotypes and determine how DRP1 and other proteins involved in mitochondrial fission/fusion affect platelet function. The results may lead to new approaches to target thrombotic disorders and enhance platelet production, leveraging a molecular target with existing inhibitors already undergoing preclinical trials for a variety of other diseases.