ANTAGONISM OF THE GPIIB IIIA RECEPTOR WITH THE NONPEPTIDIC MOLECULE BIBU52 - INHIBITION OF PLATELET-AGGREGATION IN-VITRO AND ANTITHROMBOTICEFFICACY IN-VIVO/

The glycoprotein (GP) IIb/IIIa (the alpha(IIb)beta(3) integrin) found on platelets binds fibrinogen or von Willebrand factor when the platel et is activated, thereby mediating the aggregation of platelets. Block ade of the GPIIb/IIIa should prevent platelet aggregation independent of the substance or substances responsible for activating the platelet s. This comprehensive inhibition of platelet aggregation is thought to be an effective therapeutic approach to various clinical thromboembol ic syndromes. This study investigated the platelet inhibition provided by blocking GPIIb/IIIa by using a new nonpeptidic molecule, BIBU52, i n both in vitro and in vivo models. BIBU52 competes with [I-125]fibrin ogen for binding sites on human platelets in a Ca2+ and pH-dependent m anner with a 50% inhibitory concentration (IC50) of 35 +/- 12 nM. BIBU 52 inhibited the aggregation of human platelets in platelet-rich plasm a induced by collagen (1-2 mu g/ml), adenosine diphosphate (ADP; 2.5 m u M), and a thrombin receptor-activating peptide (TRAP; SFLLRNPNDKYEPF -NH2; 25 mu M) with IC50 values of 82, 83, and 200 nM, respectively. T he inhibition of platelet aggregation by BIBU52 was found to be highly species dependent. BIBU52 inhibited aggregation in plasma from rhesus and marmoset monkeys with an IC50 Of 150 nM but was totally ineffecti ve in rat plasma. The selectivity of BIBU52 for inhibiting GPIIb/IIIa in comparison with other adhesion molecules was investigated in a huma n endothelial cell adhesion assay. The adhesion of human endothelial c ells to matrices of vitronectin, fibronectin, collagen I, or laminin w as not affected by concentrations as high as 100 mu M BIBU52; thus BIB U52 demonstrates a high selectivity for the human GPIIb/IIIa. The anti thrombotic effect of BIBU52 in vivo was investigated in three animal m odels of recurrent arterial thrombus formation. In the guinea pig aort a, BIBU52 inhibited thrombus formation dose dependently, with lack of thrombus formation for 1 h after a bolus dose of 1.0 mg/kg i.v.. Both acetylsalicylic acid and dazoxiben were less effective in this model. In pigs with recurrent thrombus formation in the carotid artery, 1.0 m g/kg i.v. also inhibited thrombus formation. Heparin was not effective in the pig, and acetylsalicylic acid was only partially effective. In the pig, the dose of 1.0 mg/kg i.v. BIBU52 also was associated with a 70% inhibition of collagen-induced platelet aggregation ex vivo but w ith only a transient prolongation of sublingual bleeding time to a max imum of 2.5-fold and without other hemodynamic effects. In the marmose t monkey, a dose of 10 mu g/kg i.v. could abolish recurrent arterial t hrombosis. Hemodynamic effects of BIBU52 in anesthetized pigs were not detected in doses less than or equal to 10 mg/kg. These data demonstr ate that BIBU52 is a potent and selective antagonist of the human GPII b/IIIa receptor and capable of substantial inhibition of platelet aggr egation in vitro and ex vivo as well as inhibition of arterial thrombu s formation in vivo in animal models of thrombosis.