Enhancement of human platelet aggregation and secretion induced by rapamycin

Background. Rapamycin is a new immunosuppressive drug of the macrolide type . Despite binding to one of the FK-binding proteins as the initial step in intracellular action, further effects differ from those of the other fungal ly derived macrolides, cyclosporine and tacrolimus. We have previously demo nstrated an enhancement of agonist-mediated platelet activation by cyclospo rine and tacrolimus which was associated with increased phosphorylation of two intracellular platelet proteins, p20 and p40. Because rapamycin utilize s the same class of binding proteins as tacrolimus, but its action is not a ssociated with the inhibition of calcineurin, we postulated that if the sti mulatory effect of cyclosporine or tacrolimus was due to calcineurin inhibi tion, rapamycin should not affect platelets in a similar fashion. Methods. Normal, washed human platelets were treated with various concentra tions of rapamycin (from ng to mu g/ml), and pre-incubated at 37 degrees C with rapamycin for various periods (1-30 min). Several platelet functional parameters were measured in samples treated with rapamycin and these parame ters were compared with control platelet samples treated with the vehicle f or the same period. Platelet aggregations following exposure to ADP or to t he thrombin equivalent, TRAP-6, were measured as changes in optical transmi ssion in a Chronolog lumi-aggregometer. Each experiment was repeated at thr ee or more times and the mean results were used for statistical comparison. Results. Rapamycin-treated platelets demonstrated an increase in their dose - and time-dependent sensitivity to ADP, resulting in a significantly enhan ced primary wave of ADP-induced platelet aggregation followed by a secondar y wave of aggregation, indicative of granule secretion. Furthermore, rapamy cin-treated platelets showed significantly enhanced sensitivity to TRAP-6 a s demonstrated by an increase in the initial velocity of aggregation, an in crease in their maximal extent of aggregation and an enhancement of granula r ATP secretion. Concentrations of rapamycin in the ng range, as well as short pre-incubatio n times (within min), were sufficient to cause significant enhancement of a gonist-induced platelet aggregation and secretion (P<0.001) as compared wit h their vehicle controls. Conclusions. Rapamycin significantly potentiates agonist-induced platelet aggregation in a time- and dose-dependent manner. As these findings are similar to those observed with the other fungal macro lides, we hypothesize that inhibition of calcineurin may not be necessary f or the increase in intracellular protein phosphorylation observed following exposure of platelets to cyclosporine or tacrolimus. Whether the rapamycin -induced enhancement of sensitivity to agonists and platelet hyperaggregabi lity explains the thrombocytopenia observed in patients when high doses of rapamycin are administered in the clinical setting, and whether these effec ts are synergistic with cyclosporine, are questions which remain to be inve stigated.