ZINC AND THROMBIN INCREASE PHORBOL ESTER BINDING TO PLATELET MEMBRANES BY A MECHANISM NOT INVOLVING TRANSLOCATION

Addition of zinc to human platelets increases the binding of phorbol e sters, and this was postulated to be the result of protein kinase C tr anslocation. To test this hypothesis, washed rat platelets were treate d with either a combination of 20 mu mol/L zinc and 20 mu mol/L pyrith ione (a zinc ionophore), or 0.1 U/mL of thrombin before determination of phorbol dibutyrate binding to subcellular fractions. As described p reviously phorbol ester binding to intact platelets was increased by t reatment with zinc and pyrithione. To obtain cytosol and mixed membran es, platelets were sonicated in a buffer containing either no chelator s or a combination of 1 mmol/Z EDTA and 2.5 mmol/L EGTA. In the absenc e of chelators, both zinc and thrombin treatment increased specific bi nding to membranes approximately 12% but had no effect on cytosol bind ing. In their presence, most binding sites were in the cytosol. Both a gonists increased membrane binding by 35% and decreased cytosol bindin g by 21%. The results suggested an increased affinity or stability of the phorbol ester binding sites associated with protein kinase C in me mbranes rather than the translocation of protein kinase C from cytosol to the membranes. To confirm that translocation was not responsible f or the increase, membranes were treated with the agonists and phorbol dibutyrate binding measured after washing with and without chelators. Binding to the washed membranes was increased approximately 20% in all cases and was generally decreased by chelator treatment. Thus, both z inc and thrombin increased binding to platelet membranes by a process that did not involve translocation.