Changes in surface-bound and exchangeable calcium during platelet activation.

Abstract

Using 45CaCl2, we measured the effect of ADP and epinephrine on exchangeable Ca2+ pools in gel-filtered platelets. Unstimulated platelets contained 1) a slowly exchangeable pool, representing 54% of the exchangeable Ca2+, that was unaffected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid and was, therefore, presumably intracellular; and 2) a rapidly exchangeable pool, representing 46% of the Ca2+, that appeared to be surface-bound because it was removable by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid or LaCl3. Equilibrium studies of this surface-bound Ca2+ revealed two classes of saturable binding sites, one class of 57,000 sites with a high affinity (KD = 5 x 10(-9) M) and another of 460,000 sites with a lower affinity for Ca2+ (KD = 3 x 10(-7) M). Two changes were observed with ADP and epinephrine. First, the amount of surface-bound Ca2+ increased by up to 50%. In the case of ADP, this included an increase in the number of lower affinity Ca2+ binding sites by 28%. Epinephrine increased the number of lower affinity sites by 18% (p less than 0.001). Second, while not increasing the amount of intracellular Ca2+, both agonists increased by 40% the rate of Ca2+ exchange into the intracellular pool. These effects were rapid (less than 1 min), dependent on agonist concentration, and inhibited by prostaglandin I2 or by specific antagonists of ADP or epinephrine binding. Thus, we find no evidence for a net influx of Ca2+ into platelets stimulated by ADP or epinephrine. However, both agonists increase the amount of Ca2+ bound to the platelet surface membrane. This bound Ca2+ may participate in several membrane reactions involved in platelet activation.