This proposal is designed to merge the candidate's background in biochemistry and clinical experience in Hemostasis and Thrombosis to produce an independent physician -scientist investigator. Platelets are critical for maintaining blood vessel integrity in response to injury, but they are also responsible for acute arterial occlusion in atherosclerotic diseases, including myocardial infarction, stroke, and peripheral vascular disease. During platelet activation, alpha granules are secreted and release important molecules for platelet function, including thrombus formation. The molecular mechanism of secretion is thought to involve interactions between alpha granule and plasma membrane SNARE proteins (N-ethylmaleimide sensitive fusion protein receptors) to form a membrane fusion complex. There are three long-term aims of this research proposal: 1. To determine the assembly, stability, and stoichiometry of the alpha granule SNARE complex; 2. To determine the membrane fusion function of the alpha granule SNARE complex; 3. To determine the high resolution X-ray crystal structure of the alpha granule SNARE complex. Secondary aims will be to determine essential SNARE sequence requirements for function, and to determine the specificity of platelet SNARE interactions compared to those in other secretory cells. The methods used to achieve these goals will include a variety of biophysical and structural techniques, including circular dichroism spectroscopy, sedimentation equilibrium analysis, fluorescence spectroscopy, and X-ray crystallography. The laboratory for this research has special expertise in studying related proteins in this way. These studies will be fundamentally important to our understanding of alpha granule secretion in platelets, and will contribute significantly to SNARE protein biology by providing insight into the specificity of membrane fusion.
