There is a fundamental gap in understanding how platelet number and function are regulated. Until we gain a more detailed understanding of this, we will lack effective therapies for patients with bleeding and clotting diseases. CMP-Neu5Ac is a sugar nucleotide that plays an important role in the biology of platelets. After release from platelets, this molecule is used as a substrate for extracellular sialylation of platelet surface proteins, resulting in changes in platelet in vivo clearance and activity. The cellular origin of the CMP-Neu5Ac released from platelets and the platelet surface protein substrates for extracellular sialylation remain unknown. The long-term goal is to capitalize on a molecular understanding of the role of platelets as mediators of extracellular sialylation to develop platelet-based cellular therapeutics for hematologic diseases and cancer. The overall objective of this application is to determine the origin of CMP-Neu5Ac in platelets and to identify its biologically relevant sialylation cosubstrates. The central hypothesis is that platelets regulate the extracellular sialylation of functionally important platelet proteins via release of CMP-Neu5Ac that is biosynthesized in platelet precursors. This will be accomplished via the following specific aims: 1) Mechanistically define the functional effects of GNE enzyme variants found in patients with inherited thrombocytopenia. This will be accomplished by overexpression and purification of the recombinant enzyme followed by in vitro enzymatic characterization. 2) Determine the cellular origin of platelet CMP-Neu5Ac. Lysates from platelets, megakaryocytes, and hematopoietic stem cells will be analyzed for expression of CMP-Neu5Ac biosynthetic enzymes, and tested for their ability to enzymatically transform UDP-GlcNAc to CMP-Neu5Ac. 3) Identify the biologically relevant platelet surface protein substrates for extracellular sialylation. Click chemistry will be used to attach alkyne-modified chemical reporters to facilitate identification of novel extracellular sialylation substrates by liquid chromatography/mass spectrometry proteomics. The outcome will be an understanding of the cellular origin of CMP-Neu5Ac in the platelet lineage and an identification of platelet surface protein substrates for extracellular sialylation. This will have implications not only for the diagnosis and treatment of disorders of hemostasis and thrombosis, but also for a broader range of diseases involving alterations of the glycome, including cancer and autoimmune diseases. The training plan includes learning a new chemical glycobiology skillset from Professor Carolyn Bertozzi at Stanford University, and applying these tools to platelet biology with guidance from consultant experts. This training will be critical for me to develop a unique niche as an independent physician-scientist investigator at the interface of chemical biology and hematology/transfusion medicine.
Platelets are critical to the pathogenesis of some of the most important causes of morbidity and mortality worldwide, including hemorrhage, heart attack, and stroke. The aim of the proposed research is to understand how a sugar molecule called CMP-Neu5Ac is made and how it is used to regulate platelet number and function. This work will illuminate fundamental aspects of platelet biology, and will pave the way for development of effective therapies for bleeding and clotting disorders.
