Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation.
The platelet integrin glycoprotein (GP) IIb/IIIa, which mediates platelet aggregation, has been the target for novel antiplatelet agents, the GPIIb/IIIa antagonists. Several GPIIb/IIIa antagonists have been developed based on the peptide RGDS present in adhesion proteins, including the principle ligand fibrinogen. The apoptosis enzyme, procaspase-3, contains an RGD-recognition sequence and is activated by RGDS. We examined the effects of RGDS and several GPIIb/IIIa antagonists on cell death and procaspase-3 activation in rat neonatal cardiomyocytes. These antagonists do not recognize rat integrins, yet RGDS, orbofiban, and xemilofiban induced dose-dependent apoptosis and procaspase-3 activation in cardiomyocytes over 72 h, particularly under hypoxic conditions. Scrambled peptide, the monoclonal antibody 7E3 or integrelin (a peptide containing a KGD sequence), had little or no effect. Immunoprecipitation of procaspase-3 followed by treatment with the compounds showed that procaspase-3 was activated directly by RGDS, orbofiban, xemilofiban, and by monoclonal 7E3 antibody, the latter demonstrating that compounds must enter cells to induce apoptosis through caspase activation. Integrelin had no effect. Binding studies with (3)H-SC52012B, a GPIIb/IIIa antagonist analogue of orbofiban, showed no specific binding to cardiomyocytes, but the radioligand accumulated intracellularly over 72 h. (3)H-SC52012B also bound directly to human recombinant caspase-3 (K(d), 59 +/- 2 nm), and this was prevented by orbofiban, xemilofiban, and the monoclonal 7E3 antibody but not by integrelin. Finally confocal microscopy showed that RGDS co-localized with caspase-3 inside the cell. These data show that RGDS and its mimetics induce cardiomyocyte apoptosis by direct activation of procaspase-3.
Adderley
S R
SR
Centre for Cardiovascular Science, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2, Ireland.
Fitzgerald
D J
DJ
D000409:Alanine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001550:Benzamidines / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000187:drug effects; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis; D000077542:Eptifibatide; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D009206:Myocardium / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors*; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014471:Umbilical Veins / Q000187:drug effects
D001550:Benzamidines; D004792:Enzyme Precursors; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011759:Pyrrolidines; C047981:arginyl-glycyl-aspartic acid; C046617:arginyl-glycyl-aspartyl-serine; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C120234:orbofiban; D000077542:Eptifibatide; D000409:Alanine; C091295:xemilofiban