In vitro effect of a thrombin inhibition peptide selected by phage display technology

• Published in: Thrombosis research Vol. 107; no. 6; pp. 365 - 371
• Main Authors: Meiring, Muriel S, Litthauer, Derek, Hársfalvi, Jolan, van Wyk, Veronica, Badenhorst, Philip N, Kotzé, Harry F
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Ltd 15-09-2002; Elsevier Science
• Subjects: Bacteriophage M13 genetics; Bacteriophages; Base Sequence; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunoassay; Medical sciences; Peptide Library; Peptides - pharmacology; Phage display system; Pharmacology. Drug treatments; Platelet Aggregation - drug effects; Receptors, Thrombin - antagonists & inhibitors; Recombinant fusion proteins; Thrombin; Thrombin antagonists and inhibitors; Thrombin Time; Peptide library; TAP; OPD; HMEC; TBS; PPACK; HRP; Thrombin; ADP; X-Gal; BSA; FITC; Thrombin antagonists and inhibitors; K i; PE; DNA; IPTG; TIP; SM; Recombinant fusion proteins; Bacteriophage M13 genetics; Phage display system; E. coli; ELISA; Human; Serine endopeptidases; Enzyme; Antithrombin; Virus; Peptidases; Platelet; Hydrolases; Inhibitor; Recombinant protein; Phage; Clone
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/S0049-3848(02)00349-3

A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the enrichment of phages that bind to human α-thrombin. One clone of the binding phages that competed with PPACK for binding to thrombin and that had the best binding characteristics was chosen. The amino acid sequence of the peptide displayed on this phage was determined and a peptide with the sequence, Cys-Asn-Arg-Pro-Phe-Ile-Pro-Thr-Cys was synthesised. This peptide, thrombin-inhibiting peptide (TIP), is a full competitive inhibitor of thrombin with an inhibition constant (
K
i) of 0.4974 mM. It lengthened the thrombin time and inhibited thrombin-induced platelet activation and the platelet release reaction, both in a dose-dependent manner. It also reduced platelet adhesion onto a human microvascular endothelial matrix in the parallel plate flow chamber under both arterial and venous shear conditions. Thus, we have selected and synthesised a cyclic heptapeptide that competes with PPACK to bind to thrombin and that can be developed as a direct antithrombin.