DESIGN AND CHARACTERIZATION OF HIRULOGS - A NOVEL CLASS OF BIVALENT PEPTIDE INHIBITORS OF THROMBIN

A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of α-thrombin. These peptides, called “hirulogs”, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 Å in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 [(D-Phe)-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu] inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with Ki = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir53-64, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with Ki = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. In addition, the pentapeptide (D-Phe)-Pro-Arg-Pro-Gly, which comprises the catalytic-site inhibitor moiety of hirulog-1, was determined to have a Ki for thrombin inhibition > 2 μM. Hirulog-1, but not S-Hir53-64, was found to inhibit the incorporation of [14C]diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitonenzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Hirulog-1 inhibited human γ-thrombin and bovine thrombin with Ki values increased 500- and 20-fold, respectively, compared to Ki for human α-thrombin. Also, hirulog-1 inhibition of α-thrombin was reversed in the presence of saturating concentrations of S-Hir53-64. Studies on the optimal length of the oligoglycyl spacer, which forms a molecular “bridge” linking active-site and ABE recognition sequences, showed that at least three to four glycines were necessary for optimal inhibitory activity. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir53-64 showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir53-64 in increasing the activated partial thromboplastin time of normal human plasma. Thus, fashioned from studies on hirudin and its fragments, synthetic peptides that bind to both the ABE and catalytic site of thrombin are potent reversible inhibitors of thrombin activities. © 1990, American Chemical Society. All rights reserved.