Inhibition of thrombosis by a selective fibrinogen receptor antagonist without effect on bleeding time
Tschopp, J.F.; Mazur, C.; Gould, K.; Connolly, R.; Pierschbacher, M.D.
Thrombosis and Haemostasis 72(1): 119-124
1994
ISSN/ISBN: 0340-6245 PMID: 7974359 Document Number: 427494
Membrane glycoprotein alpha-IIb-beta-3 on platelets plays a pivotal role in hemostasis by mediating RGD-(arginine-glycine-aspartic acid)dependent platelet adhesion and aggregation. Antagonists of alpha-IIb-beta-3 ligand binding function, such as antibodies, snake venom peptides, or synthetic RGD-containing peptides can completely inhibit platelet aggregation in vitro and cause significant prolongation of bleeding times when injected into experimental animals. The in vitro and in vivo properties of an alpha-IIb-beta-3 specific RGD-containing peptide 2G (G(Pen)GHRGDLRCA) were compared to two non-specific ROD-containing peptides 1N (G(Pen)GRGDTPCA) and 2H (GRGDSPDG). All three peptides have similar 1C-50 values in human platelet aggregation (14-22 mu-M) and ELISA-based alpha-IIb-beta-3 receptor assays (0.2-0.3 mu-M) but show different inhibitory activity (IC-50 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 receptor assays (2G = 8.3 pM; 1N = 0.06 mu-M; 2H = 0.04). The alpha-IIb-beta-3 specific peptide 2G had no effect on monolayers of human saphenous vein endothelial cells while 1N and 2H caused many cells to detach and contract. Peptides 2G and 1N inhibited ADP-stimulated ex vivo platelet aggregation in dogs in a dose dependent manner. When complete inhibition ( gt 90%) of ex vivo platelet aggregation was achieved with either a 10 mg/k- bolus followed by a 16 mg/kg/h infusion of 2G or with a 15 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 examined in a baboon ex vivo shunt model for their ability to block platelet uptake and fibrinoger deposition on small caliber GORE-TEX grafts and for their effect on the hemostatic system. Systemic administration 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 deposition on the graft surface without affecting the hemostasis and template 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 suggest that efficacy and the effect of specific alpha-IIb-beta-3 antagonists on bleeding time can be separated and that selective alpha-IIb-beta-3 receptor blockade may be an efficient and safe approach to improve the patency and the success rate of small caliber vascular grafts and to treat unwanted platelet-dependent thromboses. While peptide 2G may represent a unique class of antithrombotic agent, the clinical use of this type of molecule would require a significant enhancement in potency.