Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets.

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Abstract
We have constructed synthetic peptides modeled on the sequences of (i) Arg-Gly-Asp, present in fibrinogen, Fibronectin, and von Willebrand Factor, and of (ii) the fibrinogen .gamma. chain (.gamma. 400-411) His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val. The concentration of each peptide that inhibits 50% of 125I-labeled fibrinogen binding to thrombin-stimulated platelets (IC50) was then determined. The IC50 for (.gamma. 400-411) was 48-180 .mu.M at a fibrinogen concentration of 60 .mu.g/ml. A substitution of arginine for alanine at position 9 decreased the IC50 to 14.5 .mu.M. Arginine substitutions for all other residues on the amino-terminal side of the peptide Arg9-Gly-Asp-Val resulted in an IC50 of 0.4-0.8 .mu.M, and the IC50 of the peptide Arg13-Gly-Asp-Val was 0.2-0.3 .mu.M. This contrasts with an IC50 of 200 .mu.M for Arg5-Gly-Asp-Val-Arg4 and an IC50 > 1 mM for the peptide Arg12. The inhibitory effect resulted primarily in a decreased affinity of fibrinogen binding to platelets, although the number of available binding sites had also decreased. Binding was completely inhibited. At concentrations between 10 and 18 .mu.M, Arg9-Gly-Asp-Val blocked all ADP-induced aggregation in citrated platelet-rich plasma. The peptide Tyr-His-His-Lys-Arg-Lys-Arg-Lys-Gln-Arg-Gly-Asp-Val was labeled with 125I to quantitate its binding to thrombin-stimulated platelets; at saturation, 59,990 molecules were bound per cell (Kd = 3.8 .times. 10-7 M). These modified synthetic peptides bind to platelets with the same affinity as does intact fibrinogen and inhibit platelet function. The increased affinity of these modified peptides is > 20-fold that of peptides comprised of only native sequences and is a prerequisite for the potential antithrombotic use of these agents.

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