Abstract: Shaker A. Mousa and Thomas M. Reilly The DuPont Merck Pharmaceutical Company Cardiovascular Diseases Div., Exp. Station, Wilmington, DE 19880-0400 The present investigation was undertaken to compare the effects of different direct thrombin inhibitors including hirudin, DUP 714 (Ac-D-Phe-Pro-boro Arg) and PPACK (Phe-Pro-Arg-chloromethyl ketone) with different platelet GPIIblIIIa antagonists including linear Arginine-Glycine-Aspartate (RGD), cyclic RGD, peptidomimetic and non-peptide inhibitors on various platelet functions including: (a) platelet aggregation, (b) fibrinogen binding, (c) platelet plasminogen activator inhibitor type-l (PAl -1) release, (d) platelet serotonin release, and (e) platelet intracellular Ca++ mobilization. Hirudin, DUP 714 and PPACK were effective in inhibiting all of the responses (a-e) mediated by thrombin, with ICsos ranging from 0.002 to 0.9 uM depending upon the concentration of thrombin used, but not those mediated by other platelet agonists including collagen, ADP, and arachidonic acid. In contrast, linear RGD, constrained cyclic RGD (XM648), an RGD peptidomimetic (YY751), a non-peptide GPIIb/IIIa antagonist were effective in blocking fibrinogen binding and platelet aggregation mediated by any of the agonists. The potency of XL 111, XM648 and YY751 in inhibiting thrombin-mediated platelet aggregation (a) and fibrinogen binding (b) ranged from ICso of 0.05 to 1.0 uM in (a) and IC50 of 0.01 to 0.005 uM in (b). However, all platelet GPIIb/IIIa antagonists tested were ineffective in blocking either (c), (d) or (e). Flow cytometric analysis using dual fluorescence markers for the platelet GPIIblIIIa membrane receptors (FIrC-Iabeled cyclic RGD analog, XL086) and for the alpha granule (PE-monoclonal antibody for P-selectin) demonstrated a dissociation between the platelet GPIIb/IIIa receptors by inhibitors and platelet granular secretion. These data suggest that GPIIb/IIIa receptor blockade as compared with direct inhibition of thrombin, does not inhibit intracellular CaH mobilization signal transduction. These data also show that GPIIb/IIIa antagonists, in contrast to thrombin inhibitors, inhibit platelet aggregation mediated by different agonists. The universal anti-aggregatory efficacy of the GPIIb/IIIa antagonists and efficacy of the thrombin inhibitors against Cardiovascular Disease 2 Edited by L.L. Gallo, Plenum Press, New York, 1995 255 thrombin-induced platelet secretion suggest potential benefits of their combinations in various thrombotic disorders.