ONO-6240

The effects of platelet-activating factor (PAF) antagonists on the agonist-induced increase in cytosolic free calcium concentration, [Ca2+]i, in human vascular endothelial cells grown in monolayer were investigated by a continuous superfusion technique using a calcium fluorescent probe, fura-2. PAF caused a small but dose-dependent increase in [Ca2+]i. Seven structurally dissimilar PAF antagonists dose-dependently suppressed the peak response, among which WEB 2086 was the most potent, followed by WEB 2170 greater than FR 900452 not equal to ONO 6240 greater than BN 52021 not equal to kadsurenone not equal to CV 3988. These antagonists except for CV 3988 were specific for PAF, since they had no effects on calcium mobilization induced by thrombin or histamine, while CV 3988 had a non-specific effect. PAF in the same range of concentration increased prostacyclin release from human endothelial cells. WEB 2086 also inhibited the PAF-induced prostacyclin release, while it had not effect on the release induced by histamine and thrombin. These results demonstrate the specificity and dose-response characteristics of PAF antagonists in cultured human endothelial cells and suggest that a PAF antagonist could be a valuable therapeutic agent in certain human diseases where PAF activation of endothelial cells may have a critical role.

Platelet‐activating factor (PAF, 10 nm) did not induce platelet adhesion to endothelial cells cultured in monolayer but it induced their adhesion to protein‐coated plastic. However, PAF induced a marked platelet adhesion to endothelial cells when polymorphonuclear leukocytes (PMNs) were present. Lyso‐PAF had no effect.Phase‐contrast microscopic examination showed that single platelets rather than their aggregates adhered to the endothelial cell surface around aggregating and adhering PMNs.Significant platelet adhesion was induced by PAF at concentrations higher than 0.01 nm with the maximal response at 10 nm. Platelet adhesion occurred within minutes after PAF addition, reaching a maximum approximately after 30 min. Platelet adhesion also occurred significantly at a PMN: platelet ratio of 1:800, and linearly up to 1:50.The PAF‐induced platelet adhesion was suppressed by three structurally unrelated PAF antagonists, WEB 2086, ONO 6240 and BN 52021, in a concentration‐dependent manner.PAF also increased PMN adhesion to endothelial cell monolayers, which was further augmented by the presence of platelets.The present study demonstrates that PAF induces platelet adhesion to endothelial cells in vitro when PMNs are present and that there is a close interaction between platelets and PMNs in their adhesion to endothelial cells. The present study further suggests that PMNs could play a central role in platelet adhesion to vascular endothlium in certain pathological conditions.