EPINEPHRINE INDUCED POTENTIATION OF ARACHIDONATE AGGREGATION IN OUIN 2 LOADED PLATELETS IS NOT MEDIATED BY ELEVATION OF CYTOSOLIC CALCIUM

Previous studies have demonstrated that chelation of ionized cytosolic calcium by Ouin 2 leads to a refractory state in platelets.However, epinephrine (Epi) induced membranemodulation restored the sensitivity of Ouin 2loaded platelets to the action of agonists.Further studies with Ouin 2 and Fura 2 suggested that Epi induced recovery of sensitivity by refractory platelets to aggregation by arachidonate does not require elevation of cytosolic calcium. To further delineate the role of calcium in membrane modulation, we followed phosphoinositol metabolism and myosinlight chain phosphorylation using radiolabeled platelets. The total amount of PI metabolites generated after exposure to Epi, AA or Epi + AA were significantly less than that formed after 0.1 μ thrombin stimulation. Ouin 2 at 40 μM concentration had no inhibitory effect on PI hydrolysis. However, at this concentration it effectively blocked AA induced aggregation. Although Epi treatment restored the sensitivity of Ouin 2 loaded platelets to the action of AA, it did not enhance the formation of increased quantitites of PI metabolites. Similar to earlierstudies, Ouin 2 loading effectively blocked phosphorylation of myosin light chain (20 K).Although a combination of Epi + AA restoredsome phosphorylation of 20 K protein in Ouin2 loaded platelets, the degree of phosphorylation was significantly less than that achieved in control stimulated platelets. Resultsof these studies suggest that Epi induced restoration of sensitivity to refractory, Ouin2 loaded platelets is not mediated by 1) significant elevation of cytosolic calcium, 2) enhanced production of PI metabolites, 3) increased phosphorylation of 20 K protein. Epiinduced membrane modulation is a novel, independent mechanism capable of restoring sensitivity to agonists in platelets with compromised function.