Elevation of cytosolic calcium is thought to play a major role in platelet activation by physiologic agonists or by extended storage. The rise in cytosolic calcium induces a variety of biochemical responses including tyrosine phosphorylation and dephosphorylation of proteins. A tyrosine kinase inhibitor, Erbstatin analog, was found to selectively potentiate fibrinogen binding-mediated tyrosine phosphorylation of a single 95 kDa protein following platelet activation. This drug may be a useful tool to dissect the tyrosine kinase and phosphatases interactions which follow integrin receptor occupation and subsequent platelet aggregation. Further characterization of this protein is in progress. Current platelet storage conditions, which utilize platelet-rich plasma at room temperature, are associated with a progressive platelet activation and are a good medium for bacterial growth. Clinically, these effects can result in decreased platelet survival and the occurrence of transfusion- associated sepsis. Storage of platelets at 4 degrees C would decrease bacterial growth in contaminated platelet units and decrease the incidence of transfusion-associated sepsis, but cold-stored platelets exhibit a low degree of activation and a potentiated response to a broad range of agonists. This response involves increased expression of fibrinogen receptors without a change in their affinity for the ligand and appears to be due to changes upstream of fibrinogen binding, possibly at the level of agonist-mediated signal transduction. In the search for a synthetic storage media to extend the platelet storage period beyond 5 days, it is necessary to evaluate platelets stored in the new medium against conventionally stored platelets. We are investigating ways of decreasing the cold induced activation of platelets and evaluating platelet testing conditions, such as selection of appropriate re-suspension media after storage, so that a fair comparison of platelets stored under novel conditions could be performed. II. Hemoglobin-based blood substitutes and platelets These studies are aimed at explaining and predicting the in vivo effects of hemoglobin-based blood substitutes. Purified hemoglobin A0 (HbA0) potentiated submaximal agonist induced platelet aggregation in a concentration-dependent manner. The predominant mechanism for this potentiation was the cyclooxygenase activity of hemoglobin, which can catalyze the metabolism of arachidonic acid into prostaglandins. In addition we investigated how free radicals generated by hemoglobin could effect platelet reactivity. Peroxynitrite (ONOO), a free radical produced by the interaction of NO with superoxide, induces nitration of tyrosine residues and inhibits tyrosine phosphorylation in cell free systems. In platelets, ONOO rapidly induced tyrosine nitration of proteins and also rapidly increased tyrosine phosphorylation of a separate set of proteins. Thrombin induced the same pattern of tyrosine phosphorylation in platelets, and this was not altered by pretreatment of platelets with ONOO. Thus, tyrosine nitration does not alter thrombin induced platelet tyrosine phosphorylation, and inhibitory effects of ONOO on platelets can be independent of tyrosine nitration. We are also investigating the molecular basis of integrin aIIbb3 ligand binding by using an aIIbb3 complex-activating antibody, D3. The region of b3 integrin recognized by D3 may be an important regulatory domain in ligand-receptor interactions that directly mediate platelet aggression. These studies were also extended in a collaboration to elucidate the mechanism of thrombocytopenia associated with administration of the measles vaccine. An anti platelet antibody was found in the vaccine recipients who developed thrombocytopenia and this antibody recognized purified platelet integrin b3. The measles viral protein appears to have immunogenic homology to platelet integrin b3. III. Prion protein and Platelets The prion protein (PrPC) is expressed on many cell types and tissues and is thought to be the infectious agent in the fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE). We have investigated the role of PrPC in platelet physiology. The surface expression of PrPC, as detected by flow cytometry with a PrPC specific monoclonal antibody, increased more than twofold after platelet activation. Electron microscopy further confirmed binding of the antibody to the platelet plasma membrane. The platelet PrPC appears to be localized in a granule and is transferred to the outer plasma membrane upon activation. However, it is excluded from microparticles which are pro- coagulant pieces of membrane that are released upon platelet activation. The function of PrPC may be in mediating heterozygous platelet-cell interaction and/or adhesion.
