Zinc status and distribution of protein kinase C in rat platelets

Abstract

Platelet aggregation is impaired by zinc deficiency and in vitro zinc has been reported to affect subcellular distribution of protein kinase C (PKC), an enzyme required for platelet aggregation. In this study, the effects of zinc deprivation and in vitro calcium on phorbol ester-induced platelet aggregation and PKC distribution were investigated. Platelets were collected from rats fed a low zinc diet (<1 mg/kg) and controls that consumed a zinc adequate diet (100 mg/kg), ad libitum or pair-fed. Washed platelets were stimulated with phorbol myristate acetate (PMA, 160 nmol/L) and the rate of aggregation determined. Without added Ca 2+ the rate of aggregation was not affected by zinc status. With added Ca 2+ (1 mmol/L) the rate was decreased by zinc deficiency ( P < 0.05). For PKC measurement, platelets from each animal were pretreated briefly with either 0 or 1 mmol/L Ca 2+, then suspended in a low Ca 2+ buffer, and sonicated. Specific binding of phorbol dibutyrate (PDBu) to mixed membranes and cytosol was measured. Pretreatment with Ca 2+ increased binding to membranes and decreased binding to cytosol. Overall, zinc deficiency decreased [ 3H]PDBu binding to membranes approximately 10% ( P = 0.01), but had no effect on cytosol binding. Only in the presence of in vitro Ca 2+ did zinc deficiency decrease both PMA-induced aggregation and phorbol ester binding to mixed membranes. Zinc status had no effect on the distribution of phorbol ester binding, suggesting that low zinc status decreased availability of extracellular calcium and thus decreased membrane PKC binding affinity or the stability of PKC in the membranes