Rat platelet arachidonate metabolism in the presence of Ca 2+, Sr 2+ and Ba 2+: studies using intact platelets and semi-purified phospholipase A 2

Abstract

To document further the involvement of external Ca 2+ in the platelet-induced activation process, we have studied the arachidonate metabolism of intact washed rat platelets in the presence of different concentrations of Ca 2+, Sr 2+ or Ba 2+. The thrombin-induced mobilization of radiolabeled arachidonate preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxy genase products. Results indicate that upon thrombin stimulation (0.2 U/ml), the release of endogenous arachidonate and the formation of its metabolites are reduced by 50–90% only by omission of Ca 2+ as compared to 1 mM Ca 2+ in the suspending medium. At higher Ca 2+ concentrations (5 mM), the arachidonate mobilization and metabolite formation are inhibited and the data are thus close to those obtained in the absence of Ca 2+. In the presence of Sr 2+ or Ba 2+, the results indicate that these cations can substitute for Ca 2+. As for Ca 2+, an optimum concentration is found for Sr 2+ and Ba 2+ (3–5 mM), and higher concentrations inhibit the metabolism of arachidonic acid. As the above data might be compatible with the possible entry of Sr 2+ and Ba 2+ into platelets upon stimulation, we also studied the activity of a semi-purified preparation of phospholipase A 2 from rat platelets. This activity was assayed (pH 9.2) using heat-denatured [ 3H]arachidonate-prelabeled phospholipids as substrate. The results show that this phospholipase A 2 activity was strongly Ca 2+-dependent. In addition, we found that unlike Mg 2+, Sr 2+ and Ba 2+ are able to greatly enhance this activity. Relative efficiency ( V max ) was in the order Ca 2+ > Sr 2+ > Ba 2+. Taken together, these findings suggest that external Ca 2+ may play a major role in the regulation of rat platelet activity. Our interpretation is in line with the view that Sr 2+ or Ba 2+ could enter the platelet through a mechanism common to Ca 2+ (a Ca 2+ channel). Although direct evidence is awaited from the results of further studies which are in progress, it can reasonably be considered that Sr 2+ or Ba 2+ might cause platelet-induced activation mimicking a rise in the cytosolic Ca 2+ and subsequent activation of Ca 2+-dependent enzymes.