Calcium influx and tyrosine phosphorylation induced with thrombin are blocked by prior application of agents which elevate cAMP. It is not known whether this is due to inhibition of tyrosine kinases or activation of tyrosine phosphatases or inhibition of signal transduction such as cytosolic calcium elevation. PGE1 elevates cAMP and prevents tyrosine phosphorylation and calcium elevation but did not directly effect either tyrosine kinase or phosphatase activities. Thus, cAMP indirectly prevents agonist-induced tyrosine phosphorylation by blocking elevation of cytosolic calcium which is required for platelet tyrosine phosphorylation. Moreover, platelets release endogenous platelet agonists, ADP and thromboxane, during activation and these can potentiate platelet responses. When the release on these second agonist is blocked the tyrosine phosphorylation of specific protein becomes more sensitive to PGE1-induced elevation of cAMP. This protein is focal adhesion kinase and it appears that released ADP and cAMP antagonistically control its tyrosine phosphorylated state. We are also involved in a clinical collaboration to study platelet reactivity of patients with the Hermansky-Pudlak (HP) syndrome who lack platelet dense granules. These dense granules store calcium, ATP, ADP and serotonin and we are interested to establish the contribution of these structures to platelet activation. The first patient studied was found to be hyperresponsive to thrombin activation when platelet responses such as aggregation, cytosolic calcium elevation were tested. The HP platelets were also more sensitive to alkaline medium in that they greatly increased their volume as compared to control platelets. Since platelet volume is controlled in part by the Na+/H+ exchanger and dense granules are reported to concentrate H+, we are investigating the potential role of dense granules in regulation of intracellular pH levels and how this could increase the platelet reactivity to thrombin. The significance of these projects lies identification of the various molecular mechanisms which control platelet activation by elevation of cytosolic calcium levels as could occur during platelet storage. Inhibitors of these mechanism will be used to optimize storage conditions for platelets.
