Many systemic diseases are associated with abnormalities in platelet aggregation and storage granule secretion, but the biochemical basis for this disordered function is unknown. The major aims of these studies are to evaluate abnormalities in stimulus-response coupling of platelets taken from patients with thrombotic cardiovascular disease, to determine the effects of therapeutic interventions given to these patients on platelet function, and to assess the importance of changes in intracellular regulators to activation of normal platelets under conditions that simulate in vivo activation. Based on our previous in vitro studies demonstrating the close link between intracellular Ca++ and platelet functional changes, my current working hypotheses are that 1) a rise in cytoplasmic ionized (Ca++) in response to one agonist """"""""primes"""""""" the platelets for synergistic functional response to additional agonists and 2) that abnormalities in platelet Ca++ homeostasis may underlie functional alterations in thrombotic cardiovascular disease. Since aequorin and the fluorescent probes indo-1, quin2, and fura-2 appear to measure different aspects of Ca++ homeostasis, both will be used to record (Ca++) in platelets stimulated or inhibited by various agents. In normal platelets, the individual and net effects of subthreshold concentrations of several agonists will be compared to assess the role of Ca++ in synergy. Ca++ transients as seen with both indicators will be compared to other markers of platelet activation, including fibrinogen binding, and phosphorylation of platelet proteins, to identify physiologic correlates of increased (Ca++). Blood will also be obtained from patients with ischemic heart disease to determine if alterations in either resting or stimulated Ca++ or the relationship between Ca++ and other second messengers, are characteristic of these disorders. The Ca++ abnormalities will be compared to those found in platelets from patients with uremia, which is associated with a prolonged bleeding time for reasons that are unclear. Finally, the effect of certain therapeutic interventions on patients with ischemic heart disease will be tested, including fish oil and Ca++ antagonists, to better understand their mechanism of action. These studies should provide a greater understanding of platelet stimulus-response coupling in vivo, and may result in information of clinical importance regarding the therapeutic effects of drugs on disordered platelet function. Finally, these studies could provide the basis for the use of measurements of intracellular second messengers in clinical medicine.
