Abstract
Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.
Highlights
In hirudin derivatives was comparable in peptides of Based upon defined structural elements in reactive sites of
By comparison to unsulfated peptide, S-Hir6s-B4was found to contain sapecific inhibitory activityenhanced by one order of magnitude toward increase inAPTT and toeffect a dose-dependent increase in thrombtiinme of normal human plasma the mechanism of hirudin inhibition is unlikely to involve interaction between inhibitor and a PI reactive amino acid with the specificity pocket of thrombin
One might expect the interaction of thrombin and hirudin to be highly complex due to the high degree of specificity of hirudin for to yield a 4-fold increase in thrombitnime with 2.5 pg/ thrombin [7],the remarkable affinity of their association, and ml peptide using 0.8 units/ml a-thrombin.Comparison evidence for interaction of hirudin with low and high affinity of S-Hir63"4 to hirudin in thrombin time and Activated Partial Thromboplastin Time (APTT) sites in thrombin[5]
Summary
Structural components which mediate anti-thrombin properties of the protein We have addressed this issue by synthetic and semisynthetic approaches yielding a number of short peptide fragments based on the sequence in hirudin. Purity of synthetic peptides was routinely analyzed by reverse-phase HPLC.Peptide samples (20-100 pg)were applied to a Vydac C4 column (0.46X 25 cm) or an Aquapore RP-300 CScolumn (0.46X 3.0 cm) using a Beckman Liquid Chromatographic System or an Applied Biosystems 150-AChromatographic System, respectively.The Vydac C4column was equilibrated in water containing 0.1% trifluoroacetic acid and developed with a gradient of acetonitrile from 0 to 80% in the same trifluoroacetic acid-containing solvent over 30 min a t a flow rate of 1.0 ml/min. The column was equilibrated in water containing 0.1% trifluoroacetic acid and developed with a gradient of acetonitrile concentration from 0 to 70% over 45 min at a flow rate of 0.5 ml/min in.
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