45Ca Efflux and Agonist Induced Changes in Force in a Model of Thrombin Activated Platelets

(45)Ca(2+) and (3)H sorbitol were loaded by incubation into a model of thrombin activated, irreversibly aggregated platelets. Total Ca, measured by atomic absorption, was approximately 4.0 nmoles/mg wet weight. 55% of the total Ca(2+) was exchangeable with (45)Ca(2+), 14% was extracellular and 42% cellular, either surface or intracellular. Changes in the efflux of the marker into a buffer containing Mg-EGTA were correlated with the contractile responses of the preparation after addition of agonists. For the contracting agonists tested individually (ADP, epinephrine, and the endoperoxide analogue, U46619) the fractional efflux rate increased in phases, the descending component stabilizing at a rate higher than the basal. When agonists of different classes were added sequentially in supramaximal amounts, the increases in the stable component of the efflux were also additive and correlated well with the increases in the force of contraction. Washout of the agonist returned the efflux to the baseline. Agents increasing cyclic AMP, like prostaglandin E(1), produced a small decrease in the basal level of the efflux of (45)Ca. When contracting agonists were added to the pretreated preparation, a simultaneous decrease in efflux and force generated were found. The inhibition was dose dependent on the relaxing agonist.