Direct inhibitory mechanisms of halothane on human platelet aggregation

Background: Although halothane directly inhibits platelet aggregation, the mechanisms of this effect are still unknown. The current study aimed to clarify the inhibitory mechanisms of halothane on thrombin-induced human platelet aggregation by measuring (1) platelet-surface glycoprotein 1b expression, (2) the concentration of intracellular free Ca2+ ([Ca2+](i)) measured simultaneously with aggregation, (3) the concentration of intracellular inositol 1,4,5-triphosphate, and (4) the concentration of intracellular cyclic 3',5'-adenosine monophosphate ([cAMP](i)). Methods: Washed platelet suspensions, obtained from healthy volunteers, were preincubated with halothane (0-2 mM) for 2 min and then exposed to 0.02 units/ml thrombin for 3 min. The glycoprotein 1b bound to fluorescein- labeled antibody was measured by fluorescence flow cytometry. [Ca2+](i) was measured, simultaneously with aggregation, in Fura-2 (Ca2+ indicator)- loaded platelets by use of a fluorometer. Inositol 1,4,5-triphosphate and [cAMP](i) were measured by radioimmunoassay. Results: Halothane had no effect on glycoprotein 1b expression with or without thrombin. Halothane decreased the thrombin stimulated [Ca2+](i) transient and inhibited platelet aggregation in a dose-dependent manner, both in the presence and in the absence of external Ca2+. Isoflurane had no apparent effect on either platelet aggregation or [Ca2+](i) in the absence of external Ca2+. Halothane inhibited the increase in inositol 1,4,5-triphosphate induced by thrombin. Halothane moderately but significantly increased [cAMP](i), but the adenylate cyclase activator forskolin (which has the same inhibitory ability on aggregation as halothane) increased [cAMP](i) to a much greater extent than did halothane. Conclusions: Halothane inhibits thrombin-induced human platelet aggregation by decreasing [Ca2+](i) without inhibiting agonist- receptor binding; the inhibitory effect of halothane on [Ca2+](i) might be mediated by a decrease in inositol 1,4,5-triphosphate and in part by an increase in [cAMP](i).