Revised Manuscript Received April 23, 1990 abstract: 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 a-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 i 8 A 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 K {= 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 K {= 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-siteinhibitor moiety of hirulog-1, was determined to have a K; for thrombin inhibition> 2 jiM. 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 optimalinhibitory activity of hirulog-1 depends upon all three components of its structure. Hirulog-1 inhibited human 7-thrombin and bovine thrombin with K {values increased 500-and 20-fold, respectively, compared to Kt for human a-thrombin. Also, hirulog-1 inhibition of a-thrombin was reversed in the presence of saturating concentrations of S-Hirj^. 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-Hi^^ 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 thrombinactivities. e interaction of thrombin with macromolecular substrates, inhibitors, and cofactors involves multiple, discrete structural regions that include the catalytic site, substrate groove, and anion-binding exosite, ABE1 (Fenton, 1986; Fenton et al., 1988).

The involvement of such multiple structures in the association of thrombin with macromolecules may be key in governing specificity of this central bioregulatory enzyme of thrombosis and hemostasis. Hirudin, a natural inhibitor of thrombin, appears to exploit these structural principles in formation of a tight stoichiometric complex with enzyme. The inhibition of thrombin by hirudin has been shown to involve interactions at both a “high-affinity”(Kd= 10~ 8 M), noncat-alytic site and a second site at or near the catalytic triad (Stone & Hofsteenge, 1986; Fenton et al., 1990). We have shown that a Tyr-sulfated dodecapeptide derived from residues 53-64 of hirudin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a potency in plasma reduced by