The overall quality of platelets for transfusion is not considered satisfactory by most workers in the field. The problem is due partly to the inherent difficulty of collecting and storing these highly reactive cells, and partly to the lack of adequate in vitro assay methods. The techniques available in modern cell biology have not been applied fully to investigation of the cellular and biochemical basis of the platelet storage lesion. We have available several very sensitive methods for identifying storage-induced changes in the platelet membrane, the membrane skeleton and the cytoskeletal proteins. Characterization of protein patterns has been achieved by one-and two dimensional SDS-polyacrylamide gel electrophoresis under both non-reducing and reducing conditions. Identification of proteins is performed by immunoblotting using a variety of monoclonal antibodies to actin, myosin, actin-binding protein and tubulin and antibodies to membrane glycoproteins lb, llb llla and the llb/llla complex. Activation of platelets during storage is being investigated by flow cytometry with an antibody to padgem (platelet activation dependent granule-external membrane protein). A method to analyze activation-dependent changes in platelets sampled directly form the platelet concentrates has been developed and includes formalin- fixation of platelets diluted in plasma with formaldehyde prior to staining with fluorescein-labelled antibodies. Washing steps have been eliminated from this procedure, allowing platelets to be analyzed directly without centrifugations which may cause additional activation. In addition, the amount of filamentous actin, also indicative of activation, will be measured with the sensitive probe, rhodamine- labelled phalloidin. We will correlate the activation state data with morphological alterations by phase microscopy. Results of a study of cytoskeletal changes occurring in platelets stored for 5 days were presented in a major symposium entitled """"""""The Cellular and Molecular Basis of the Platelet Storage Lesion"""""""" which was held in April, 1991.
