Chemical Synthesis of Echistatin, a Potent Inhibitor of Platelet Aggregation from Echis carinatus: Synthesis and Biological Activity of Selected Analogs

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-06, Vol.86 (11), p.4022-4026
• Main Authors: Garsky, Victor M., Lumma, Patricia K., Freidinger, Roger M., Pitzenberger, Steven M., Randall, William C., Veber, Daniel F., Gould, Robert J., Friedman, Paul A.
• Format: Article
• Language: English
• Subjects: Aggregation; Amino Acid Sequence; Amino acids; Biochemistry; Biological and medical sciences; Blood coagulation. Blood cells; Circular Dichroism; Echis carinatus; Fundamental and applied biological sciences. Psychology; Humans; Indicators and Reagents; Inhibitory concentration 50; Molecular and cellular biology; Molecular Sequence Data; Oxidation; Peptides; Platelet; Platelet aggregation; Platelet Aggregation Inhibitors - chemical synthesis; Platelets; Protein Conformation; Protons; Resins; Solvents; Structure-Activity Relationship; Viper Venoms - chemical synthesis; Viper Venoms - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.11.4022

Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50= 3.3 × 10-8M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50= 1.05 × 10-7M. Substitution with Ala-24 gave IC50= 6.1 × 10-7M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50= 6 × 10-6M), Orn-Gly-Asp-Phe (IC50= 1.3 × 10-4M), and Ala-Gly-Asp-Phe (IC50= 5.0 × 10-4M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin.