Effect of Ca2+ chelation on the platelet inhibitory ability of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban

Objective. Enhanced GPIIb/IIIa binding and inhibition of platelet aggregati on of eptifibatide by the reduction of ionized plasma calcium concentration s have been reported. The present study compared the importance of Ca2+ che lation on the in vitro platelet inhibitory profiles of the GPIIb/IIIa antag onists abciximab, eptifibatide and tirofiban. Methods and Results. Turbidim etric platelet aggregation dose response curves of the various GPIIb/IIla a ntagonists were performed using platelet rich plasma (PRP) anticoagulated w ith either trisodium citrate, or the non-chelating anticoagulant, PPACK, Th e concentrations of antagonist that resulted in 50% inhibition of TRAP-indu ced (10 muM) platelet aggregation (IC50) were measured in the presence of e ither citrate or PPACK. In addition, the influence of Ca2+ chelation on the binding properties (relative affinity, on- and off-rates) of abciximab for the GPIIb/IIIa receptor on platelets was measured. For all three agonists, the IC50 concentrations were lower for platelets treated with citrate than PPACK, but the degree of difference varied among the agents. The mean TRAP IC50 Values for citrate and PPACK were 88.2 +/- 12.2 nM and 126.1 +/- 28.4 nM for abciximab (1.4 fold enhancement; p = 0.0007), 75.9 +/- 13.3 nM and 142.6 +/- 32.6 nM for tirofiban (1.9-fold enhancement; p = 0.001), and 260. 2 +/- 62.5 nM and 810.3 +/- 182.5 nM for eptifibatide (3.1-fold enhancement ; p = 0.001). A similar shift in effective inhibitor concentrations for abc iximab was observed with ADP (10 muM). The relative affinities (EC50), on- and off-rates of abciximab for the platelet GPIIb/IIIa receptor in the pres ence of trisodium citrate and PPACK were equivalent. Conclusions. These dat a confirm previous observations that Ca2+ chelation afforded by citrate dec reases the effective inhibitor concentrations of GPIIb/IIIa antagonists, as assessed by turbidimetric platelet aggregation. However, the extent of dec rease was less for abciximab and tirofiban, compared to eptifibatide.