The proposed program is focused on elucidation of the kinetics and mechanisms of platelet response in a shear field. Human blood platelets will be studied in whole blood and in a number of different suspensions with and without added agonists, subjected to measured, known levels of shear stress, and examined for evidence of response. Rotational viscometers will be used so that a very wide range of stress levels and exposure times can be studied under closely controlled conditions. Basic indices of platelet response will include aggregation in the shear field, release and functional changes. Most of the emphasis will be on other indicators of platelet response as outlined below. A rotational viscometer will be fitted with optical detection systems so that platelet response can be monitored directly in the shear field. Three indicators of platelet response will be monitored: (1) aggregation, indicated by optical density changes; (2) dense granule release, followed by measuring ATP - luciferin - luciferase luminescence; and (3) intracellular calcium ion mobilization, followed through use of photoprobes. The influence of exposure to shear stress on rates of arachidonic acid (AA) uptake will be established. Measurements will be made of the distribution amongst phospholipids, neutral lipids, and free fatty acids. Metabolites of AA will be measured and platelet receptor changes will be studied both directly and indirectly. Platelet receptor glycoproteins will be studied by use of monoclonal antibodies directed against specific platelet receptors. Other studies will determine the influence of the shear field on the attachment to platelets of the vWF (von Willebrand Factor) multimeric forms in normal plasma, and the unusually large vWF multimers produced and secreted by human endothelial cells.
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