EFFECT OF CA2-IIIA INTERACTIONS WITH INTEGRILIN - ENHANCED GP IIB-IIIA BINDING AND INHIBITION OF PLATELET-AGGREGATION BY REDUCTIONS IN THE CONCENTRATION OF IONIZED CALCIUM IN PLASMA ANTICOAGULATED WITH CITRATE( ON GP IIB)

Background Integrilin (eptifibatide), a potent inhibitor of the fibrin ogen binding function of GP IIb-IIIa, has been shown to reduce the thr ombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca2+ c oncentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements fo r this drug. Methods and Results Lower concentrations of Integrilin we re found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean+/-SD IC50=140+/-40 nmol/L n=6; Ca2+=40 to 50 m u mol/L) than with PPACK (IC50=570+/-70 nmol/L, P<.0001, n=6; Ca2+ app roximate to 1 mmol/L). Chelation of Ca2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrili n. Measurements of D3 LIES epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by I ntegrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that m imics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca2+ als o increased Integrilin inhibition of the binding of biotinylated fibri nogen to purified, immobilized GP IIb-IIIa. Conclusions These data sug gest that citrate anticoagulation removes Ca2+ from GP IIb-IIIa and en hances the apparent inhibitory activity of Integrilin. This finding in dicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possi ble to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.