Search

1 We have previously demonstrated that nitric oxide (NO) triggers CD34(+)-derived megakaryocyte apoptosis. We here show that prostacyclin (PGI(2)) inhibits PAPA/NO-induced megakaryocyte death detected by fluorescent microscopy and flow cytometry. 2 The cAMP-specific phosphodiesterase inhibitor, Ro 20-1724, and the permeable analog dibutyryl-cAMP also delayed apoptosis. PGI(2) effect was fully prevented when adenylyl cyclase activity was suppressed by SQ 22536, and partially reversed by the permeable protein kinase A inhibitor PKI 14-22 amide. ELISA showed that while both PGI(2) and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels. 3 Treatment of megakaryocytes with PGI(2) abolished both basal and NO-raised cGMP levels. Addition of 8-pCPT-cGMP or activation of soluble guanylyl cyclase by BAY 41-2272 induced cell death in a concentration-dependent manner, and ODQ, an inhibitor of guanylyl cyclase, prevented both PAPA/NO- or BAY 41-2272-induced apoptosis. Specific cGMP phosphodiesterase inhibition by Zaprinast or suppression of adenylyl cyclase by SQ 22536 enhanced the PAPA/NO proapoptotic effect. 4 PGI(2) completely inhibited NO-mediated generation and the increased activity of the cleaved form of caspase-3. 5 In conclusion, our results demonstrate that contrary to their well-known direct and synergistic inhibitory effects on platelets, PGI(2) and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase-3 activity control.

1. The effect of unstimulated human polymorphonuclear leukocytes (PMNs) on platelet activation was examined. 2. Human platelet aggregation and adenosine 5'-triphosphate (ATP) release induced by collagen (1-2 micrograms ml-1); thrombin (0.01-0.02 u ml-1) or arachidonic acid (AA) (0.1-0.2 mM) were markedly inhibited when conducted in the presence of unstimulated PMNs. 3. Platelet inhibition induced by PMNs was dependent on the number of PMNs and on the incubation time of the mixed cell suspension. 4. Platelet inhibition was not reversed in time when PMNs were depleted from the mixed-cell suspension. 5. PMN-mediated platelet-inhibition was not mediated by AA metabolites, oxygen reactive intermediates, nitric oxide or proteases. 6. The factor(s) accounting for the platelet inhibition mediated by PMNs are not yet characterized.

1. Two non-lipid antagonists of platelet-activating factor acether (Paf), BN 52021 and WEB 2086, at concentrations which completely blocked Paf-induced platelet aggregation, failed to interfere with aggregation by adrenaline. In contrast, Ro 19-3704, a structurally related antagonist of Paf, inhibited concentration-dependently aggregation induced by adrenaline or by the simultaneous addition of submaximal concentrations of adrenaline and Paf. Reversal of aggregation was obtained when Ro 19-3704 was added to the platelet suspension after adrenaline. 2. Ro 19-3704 was selective for Paf and adrenaline since it failed to interfere with platelet aggregation induced by arachidonic acid or ADP. CV-3988, an antagonist of Paf structurally similar to Ro 19-3704, also inhibited adrenaline-induced aggregation. However, a morpholine analogue (MA) of Paf, which has no anti-Paf activity, failed to interfere with the aggregation induced by adrenaline. This suggests that the effect of Ro 19-3704 and CV-3988 on adrenaline is not simply due to their lipid structure. 3. Experiments on plasma membrane preparations showed that Ro 19-3704 inhibited [3H]-yohimbine binding with an inhibition constant (Ki) of 7 +/- 3 microM. In contrast, BN 52021 and MA did not interfere with [3H]-yohimbine binding. Equilibrium binding experiments showed that Ro 19-3704 increased the apparent KD of [3H]-yohimbine binding from 2.02 +/- 0.15 to 7.3 +/- 0.4 nM. The Paf antagonist Ro 19-3704 interacts specifically with the alpha 2-adrenoceptor and may thus prevent the early steps involved in the mechanism of adrenaline-induced platelet activation.