Beta-lactam antibiotics exert inhibitory effects on platelets, in part, by binding to the platelet surface and thereby preventing interaction of platelet agonists with their receptors. We are investigating the effect of antibiotics such as penicillin on platelet membrane glycoproteins which serve as receptors for agonists such as thrombin and ADP and as binding sites for von Willebrand Factor and fibrinogen. A study of structural changes in platelet membrane glycoproteins, as well as in membrane skeleton proteins and cytoskeleton proteins, has been initiated. Purification methods have been optimized for the isolation of platelet membranes and subcellular fractions from freshly collected platelets. Characterization of proteins has been achieved by one-dimensional SDS-polyacrylamide gel electrophoresis under both non-reducing and reducing conditions. Identification of proteins is currently performed by immunoblotting using a variety of monoclonal antibodies to glycoproteins Ib, IIb, IIIa, and the IIb/IIIa complex and the cytoskeletal proteins actin, myosin, actin-binding protein and tubulin. Dot-blotting of platelet protein fractions was employed In order to establish tiers of these antibodies. In addition, we have recently acquired an antibody which Is directed towards a glycoprotein expressed on the surface of activated platelets (anti-PADGEM, platelet activation dependent granule-external membrane protein) which will serve as a sensitive probe of the activation state of antibiotic-treated platelets. This study will be expanded to include an investigation of protein changes in platelets stored under blood banking conditions for up to 5 days and treated with antibiotics or anti-platelet drugs. We will correlate immunochemical with morphological alterations using phase microscopy and the activation state of platelets by flow cytometry with anti-PADGEM antibody.
