Thrombin specificity

Abstract

a-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative aminoterminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of a-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by a-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit a-thrombin in 20 pl of 50 mM ammonium bicarbonate at 25 "C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for a-thrombin has the structures of (a) P4-P3-Pro-Arg-P 1'-P2', where P3 and P4 are hydrophobic amino acid and PI', P2' are nonacidic amino acids and (b) P2-Arg-P l', where P 2 or P 1' are Gly. The requirement for hydrophobic P 3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P 3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P 1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by a-thrombin can be SedSOnably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage. a-Thrombin is a trypsin-like serine proteinase which preferentially cleaves polypeptide substrates at Arg/Lys-Xaa bonds [I -31. Unlike trypsin, a-thrombin displays highly restricted specificity toward protein substrates. This specificity is best known by its selective cleavage of two Arg-Gly bonds from amongst 181 Arg/Lys-Xaa bonds in fibrinogen [4]. Attempt to delineate the structural element in fibrinogen which accounts for restricted thrombin cleavage has mostly focussed on the fibrinogen Aa chain [5 -81 and has led to the findings that (a) the Aa (1-23) fragment contains a fundamental structure element for thrombin binding Table 1 for numbering of residues.) The recognization of the importance of hydrophobic P2 and P9 residues has further led to the development of a series of useful tripeptide substrates [9 -131 and inhibitors [I 4, 151. Another source of understanding of thrombin specificity comes from thrombin cleavage at non-fibrinogen polypeptides. A number of peptides and proteins have been Abbreviations. DABS-GI, dimethylaminoazobenzene sulfonyl chloride;...

Biologically Active Recombinant Human Progastrin6–80Contains a Tightly Bound Calcium Ion

“…This peptide would be generated by cleavage between Arg 5 and Ser 6 in the progastrin sequence Trp-Lys-Pro-Arg-Ser-Gln, which is consistent with the preferred thrombin recognition sequence P4-P3-Pro-Arg/Lys-P1Ј-P2Ј, where P3 and P4 are hydrophobic amino acids, and P1Ј and P2Ј are nonacidic amino acids (27). Isolation of progastrin and progastrin 6 -35 from a human gastrinoma by Reeve and co-workers (28) had previously revealed that the signal peptide of preprogastrin was 21 amino acids long and that an additional cleav- .…”

Proteolytic inactivation of ADAMTS13 by thrombin and plasmin

“…31,32 Following the initiation of coagulation, thrombin confers its prothrombotic activity by cleaving fibrinogen, feedback activation of up-stream clotting factors, and the activation of tethered platelets. 33 Together, these functions promote rapid thrombus formation.…”

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains

“…These GST-toxin fusion proteins were purified by affinity chromatography on glutathione agarose under non-denaturing conditions. The vector also encodes a thrombin recognition site (L-V-P-R-G-S) which allows the separation of the GST and toxin polypeptides by endopeptidolytic cleavage with thrombin (Chang, 1985). The vector pGEX-KG is a derivative of the vector pGEX-2T (Pharmacia), which has a glycine-rich linker incorporated adjacent to the thrombin site (Guan & Dixon, 1991).…”