Runx1 Haplodeficiency, Endocytosis and Vesicle transport

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Project Summary RUNX1 is a major hematopoietic transcription factor and RUNX1 haplodeficiency (RHD) is characterized by familial thrombocytopenia and impaired platelet function. We have a longstanding interest in the molecular basis of inherited platelet dysfunction, particularly related to RHD. Numerous platelet abnormalities have been described in RHD, several by us, and include deficiencies of dense granule (DG) and α-granules (AG). Our platelet expression profiling studies in a patient with RHD showed that several genes are down regulated (MYL9, PRKCQ, ALOX12, PF4, PLDN, PCTP); and we have shown that these are direct RUNX1 transcriptional targets and impact platelet/megakaryocyte (MK) biology. The overall objective of this proposal is to obtain new insights into the mechanisms of endocytosis, vesicle trafficking, and α-granule formation in platelets/MK through the study of phenotypic abnormalities and genes dysregulated in RHD. This project builds on unique abnormalities identified by us in RHD. We have reported in our patient that platelet albumin and IgG (incorporated by bulk endocytosis into AG) are decreased. Our expression profiling studies show that platelet RAB1B, RAB31 and DNM3 – three GTPases closely linked to vesicle trafficking are decreased. Little is currently known regarding the mechanisms regulating endocytosis in platelets/MK or the role of these GTPases. Our hypothesis is that mechanisms of endocytosis and vesicle trafficking are impaired in RHD. Aim 1 is to obtain insights into mechanisms regulating endocytosis in platelets/MK through the study of mechanisms leading to the decreased platelet albumin and IgG in RHD. We will study uptake and transport of albumin and IgG in normal and RHD platelets, study the effect of downregulation of RUNX1, RAB1B, RAB31 and DNM3 on these processes in MK, perform studies using markers of secretory and endocytic pathways of vesicle trafficking. Aim 2 is to understand the mechanisms leading to AG deficiency in RHD. We will study: platelet AG in normal and RHD platelets focusing on selected AG proteins; the effect of downregulation of RUNXI, RAB1B, RAB31and DNM3 on AG and trafficking of AG proteins in MK. These studies will be performed on 5-6 patients with RHD and using induced pluripotent stem cells (IPSCs) already developed from a RHD patient. They will provide important new insights into endocytosis and vesicle trafficking in platelets/MK, about which little is presently known. Relevance Platelets play a major role in hemostasis, thrombosis, inflammation, atherosclerosis and handling of pathogens. Our studies will provide new information on the basic aspects of platelet/MK function through studies in human RUNX1 haplodeficiency, a unique reservoir of information. This information will lay the foundation for new therapeutic approaches for both thrombotic and bleeding disorders.