INHIBITION OF THROMBOSIS BY A SELECTIVE FIBRINOGEN RECEPTOR ANTAGONIST WITHOUT EFFECT ON BLEEDING-TIME

Membrane glycoprotein alpha(IIb)beta(3) on platelets plays a pivotal r ole in hemostasis by mediating RGD-(arginine-glycine-aspartic acid)-de pendent platelet adhesion and aggregation. Antagonists of alpha(IIb)be ta(3) ligand binding function, such as antibodies, snake venom peptide s, or synthetic RGD-containing peptides can completely inhibit platele t aggregation in vitro and cause significant prolongation of bleeding times when injected into experimental animals. The in vitro and in viv o properties of an alpha(IIb)beta(3) specific RGD-containing peptide 2 G (G(Pen)GHRGDLRCA) were compared to two non-specific RGD-containing p eptides 1N (G(Pen)GRGDTPCA) and 2H (GRGDSPDG). All three peptides have similar IC50 values in human platelet aggregation (14-22 mu M) and EL ISA-based alpha(IIb)beta(3) receptor assays (0.2-0.3 mu M) but show di fferent inhibitory activity (IC50 values) in the alpha(v) beta(5) (2G = 10 mu M; 1N = 0.06 mu M; 2H = 0.05 mu M) and alpha(5) beta(1) recept or assays (2G = 8.3 mu M; 1N = 0.06 mu M; 2H = 0.04). The alpha(IIb)be ta(3) specific peptide 2G had no effect on monolayers of human sapheno us vein endothelial cells while 1N and 2H caused many cells to detach and contract. Peptides 2G and 1N inhibited ADP-stimulated ex vivo plat elet aggregation in dogs in a dose dependent manner. When complete inh ibition (>90%) of ex vivo platelet aggregation was achieved with eithe r a 10 mg/kg bolus followed by a 16 mg/kg/h infusion of 2G or with a 1 5 mg/kg bolus and 24 mg/kg/h infusion of 1N, peptide 1N caused a dose- dependent increase of the template bleeding time, while peptide 2G had no effect, even at doses up to 15 mg/kg bolus followed by 24 mg/kg/h infusion. The in vivo properties of peptides 2G and 2H were also exami ned in a baboon ex vivo shunt model for their ability to block platele t uptake and fibrinogen deposition on small caliber GORE-TEX (R) graft s and for their effect on the hemostatic system. Systemic administrati on of peptide 2G at 10 mg/kg bolus followed by 10 mg/kg/h infusion (or at a 2-fold lower dose) abolished platelet uptake and fibrinogen depo sition on the graft surface without affecting the hemostasis and templ ate bleeding time of the animal. By contrast, peptide 2H caused a 3-4- fold increase in bleeding time at a dose of 10 mg/kg. The results sugg est that efficacy and the effect of specific alpha(IIb)beta(3) antagon ists on bleeding time can be separated and that selective alpha(IIb)be ta(3) receptor blockade may be an efficient and safe approach to impro ve the patency and the success rate of small caliber vascular grafts a nd to treat unwanted platelet-dependent thromboses. While peptide 2G m ay represent a unique class of antithrombotic agent, the clinical use of this type of molecule would require a significant enhancement in po tency.