Antithrombin properties of C-terminus of hirudin using synthetic unsulfated Nα-acetyl-hirudin45–65

AbstractUnsulfated Nα-acetyl-hirudin45–65 (MDL 27 589), which corresponds to the C-terminus of hirudin1–65, was synthesized by solid-phase methods. The synthetic peptide was able to inhibit fibrin formation and the release of fibrinopeptide A from fibrinogen by thrombin. The catalytic site of thrombin was not perturbed by the synthetic peptide as H-D-Phe-Pip-Arg-pNA hydrolysis (amidase activity) was not affected. The binding of synthetic peptide and thrombin was assessed by isolation of the complex on gel-filtration chromatography. A single binding site with a binding affinity (Ka) of approx. 1.0 × 105 M−1 was observed for thrombin-hirudin45–65 interaction. The data suggest that the C-terminal residues 45–65 of hirudin contain a binding domain which recognizes thrombin and yet does not bind to the catalytic site of the enzyme

Probing Proteinase Active Sites Using Oriented Peptide Mixture Libraries – ADAM-10

Oriented Peptide Mixture Libraries can provide a full matrix of preferred and disfavored amino acids at each subsite of an optimal substrate for a new proteinase. This approach is rapid and convenient, requiring only two mixture libraries to complete the analysis. In this paper we demonstrate an extension of this type of analysis, using a focused library employing unnatural amino acids to probe the depth of the S1 position in the catalytic site of the alpha secretase ADAM-10. This analysis indicates that ADAM- 10 will accept amino acids with substantial length and hydrophobicity (e.g. 2- naphthylalanine), but suggests that the S1 site has limitations in the apparent “width” of substituents being presented (e.g. 1-naphthylalanine; gamma branching). A highly selective and efficient substrate for ADAM-10, with a selectivity factor of 380,000 M-1 s -1 , was derived from the predicted consensus substrate. This detailed analysis provides a starting point for the design of inhibitors of this interesting proteinase

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93–119 to sulfatide (Gal(3-SO4)β1-1Cer) by glycosaminoglycans

AbstractLeech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)β1-1Cer)and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93–119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 ≅ dextran sulfate 500 0OO > heparin > dermatan sulfate ⪢ chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93–119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids