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We evaluated the characteristics of the protein kinase C (PKC)-independent mechanism for ATP release in platelet-rich plasma. When ADP (10 microM) and U46619 (1 microM) were both added as agonists, a significant release was observed immediately after stimulation. The PKC inhibitor, Ro-31-7549 (10 microM), or a cyclooxygenase inhibitor, aspirin (400 microM) or indomethacin (20 microM), partially inhibited ATP release with little effect on platelet aggregation. The ATP release observed in the presence of Ro-31-7549 was abolished by a cyclooxygenase inhibitor or by preventing aggregation without stirring. In the nonstirred condition, thromboxane B2 formation was reduced by 93%. When sodium arachidonate (1 mM) rather than U46619 was used with ADP, ATP release in the presence of Ro-31-7549 was abolished by stopping the stirring with no effect on thromboxane B2 formation. In contrast, ADP/U46619-induced ATP release observed in the presence of aspirin was only partially inhibited when the stirring was stopped. This release was also inhibited dose-dependently by Ro-31-7549 at concentrations between 1 and 10 microM. These results suggest that PKC-independent ATP-release in this system requires aggregation and is inhibited by a cyclooxygenase inhibitor, while PKC-dependent exocytosis is insensitive to aggregation and a cyclooxygenase inhibitor.

We examined the roles of myosin light-chain kinase in platelet responses to ADP using wortmannin, which almost completely inhibited myosin light-chain kinase at 3-6 microM. This concentration of wortmannin did not affect ADP-induced changes in the shape of the platelets, but it markedly inhibited aggregation in platelet-rich plasma and washed platelets. ML-9, another inhibitor of myosin light chain kinase, elicited similar effects on the platelet responses to wortmannin. Electron microscopic studies showed that there was no wortmannin effect on the ADP-induced spheration of discoid platelets, pseudopod formation, or granule centralization. Wortmannin at concentrations which prevented myosin light-chain kinase also inhibited platelet aggregation induced by ADP in the presence of U46619, an analogue of thromboxane A2, which is a prerequisite for ADP-induced irreversible aggregation. Although wortmannin partially inhibited protein kinase C, the protein kinase C inhibitor Ro-31-7549 (5 microM) prevented neither ADP- or ADP/U46619-induced changes in the shape of the platelets nor aggregation. These results suggest that myosin light-chain kinase activation is a prerequisite for ADP-induced platelet aggregation, but not for changes in their shape.

We examined the mechanisms of ATP release by human platelets using Ro-31-7549, a specific inhibitor of protein kinase C. Ro-31-7549 almost completely inhibited TPA-induced platelet aggregation and ATP release at 5-10 microM in washed platelets and in platelet-rich plasma. However, it suppressed thrombin- and U46619-induced ATP release by only 48% and 21%, respectively, and had little effect on aggregation in washed platelet suspensions containing serum or in platelet-rich plasma. The addition of GRGDS to prevent aggregation inhibited this residual thrombin-induced release by 53% and the residual U46619 release by 100% in the presence of Ro-31-7549. In washed platelet suspensions free of serum or plasma, Ro-31-7549 almost completely inhibited the ATP release and partially suppressed the aggregation induced by these agonists. These results suggested that there are protein kinase C-dependent and -independent mechanisms for ATP release by human platelets and that activation of the latter mechanism may depend on aggregation and plasma factors.

Thapsigargin-activated Ca2+ entry into platelets was examined in the presence of S-145, a thromboxane A2 receptor antagonist, to inhibit indirect effects by endogenously formed prostaglandin H2/thromboxane A2. With external Ca2+ present, 0.2 microM thapsigargin caused a prompt increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a gradual increase. Pretreatment with 6 microM wortmannin, a specific inhibitor of myosin light chain kinase, partly inhibited the increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thapsigargin induced a smaller increase in [Ca2+]i, and subsequent addition of Ca2+ to the buffer caused a further prompt increase in [Ca2+]i, demonstrating external Ca2+ entry. Wortmannin only partly inhibited this entry of external Ca2+. The wortmannin-insensitive Ca2+ entry pathway remained open for more than 6 min in Ca(2+)-free buffer. On the other hand, when receptor agonists such as thrombin and U46619 were substituted for thapsigargin, activation of the wortmannin-insensitive Ca2+ entry was transient (Hashimoto et al., J. Biol. Chem (1992) 267, 17078-17081). In the presence of S-145 and wortmannin, thapsigargin stimulated phosphorylation of neither the 20-kDa myosin light chain nor the 47-kDa protein, a substrate of protein kinase C. These results suggest that thapsigargin induces external Ca2+ entry by two mechanisms: (1) a mechanism involving myosin light chain kinase; (2) a mechanism, not activated by receptor agonists, that is independent of the major protein kinases of platelets.