Studies on the binding of H-3-SR121566, an inhibitor of Gp IIb-IIIa activation

SR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. H-3-SR121566 bound with n anomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing c ells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 t o platelets, was displaced by several agents including RGD-containing pepti des and synthetic RGD mimetics, but not by ReoPro(R), a humanised monoclona l antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa comp lex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able t o compete with SR121566 whether platelets were activated or not. Flow cytometry studies indicated that SR121566 which did not activate Gp II b-IIIa by itself, dose-dependently prevented the detection of activation-in duced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation stat e of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the ac tivation of Gp IIb-IIIa and to displace the binding of fibrinogen when adde d up to 5 min after TRAP stimulation of platelets. When added at later time s (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if S R121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated. In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptid e sequence present on its gamma chain, and that this interaction is inhibit ed by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.