PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how platelet clearance is regulated. Until we gain a more detailed understanding of this, we will lack effective therapies for some patients with low platelet counts (thrombocytopenia) who are at risk for life-threatening bleeding. One important trigger for platelet clearance from the body is desialylation, which refers to the removal of the sugar molecule sialic acid from glycoproteins on the surface of the platelet. Platelet desialylation plays a role in accelerated clearance of platelets in immune thrombocytopenia (ITP) and following transfusion of platelets that have been stored at cold temperatures. Despite the clinical importance of desialylation, both the identity of the enzyme that cleaves platelet sialic acid and its scope of glycoprotein substrates and products remain unknown, hampering efforts to develop targeted therapies for ITP and other thrombocytopenic disorders. The central hypothesis is that human neuraminidase 1 desialylates glycoprotein (GP) Ibα O-glycans as well as other platelet surface glycoproteins, thereby accelerating platelet clearance in ITP and after cold storage. This hypothesis will be tested via the following specific aims: 1) Determine which GpIbα glycans undergo desialylation. Liquid chromatography/mass spectrometry (LC/MS) will be used to analyze GpIbα purified from platelets after desialylation is triggered in vitro either by incubation at 4°C or by incubation with sera from ITP patients containing anti-GpIbα autoantibodies. 2) Determine which human neuraminidase desialylates glycans that are relevant for platelet clearance. The effect of a panel of potent and selective small molecule neuraminidase isoenzyme inhibitors will be tested in in vitro platelet desialylation experiments. The in vivo half life of platelets treated with these desialylation inhibitors will be measured. 3) Identify platelet neuraminidase substrates. An established strategy for enrichment of membrane glycoproteins will be employed to allow LC/MS proteomics analysis of desialylated vs control platelets. The outcome will be a delineation of the key platelet neuraminidase enzyme which catalyzes desialylation and its glycoprotein substrates and products. This will provide the first biochemical characterization of the process of platelet desialylation and will pave the way for development of more effective therapies for thrombocytopenia. The career development plan includes 1) training in chemical glycobiology as a member of the laboratory of Prof. Carolyn Bertozzi at Stanford University and 2) training in platelet biology, mouse platelet transfusion experiments, and immune thrombocytopenia under the mentorship of international experts at Stanford and elsewhere, including Drs. Karin Hoffmeister, David Kuter, and Lawrence Leung. This will position Dr. Hollenhorst to establish a unique niche as an independent physician-scientist investigator at the interface of chemical biolo...