The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1
Research article published in European journal of biochemistry (2001)
Abstract
The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis.
Why This Matters for Hirudotherapy
This study used site-directed mutagenesis of thrombin's anion-binding exosite-I (ABE-I) basic residues to dissect their roles in recognizing and cleaving protease-activated receptor 1 (PAR1). Mutations of Arg67 and Arg73 most dramatically reduced PAR1 peptide cleavage efficiency (kcat/Km dropping from 9.2×10^7 to 1.8×10^6 and 4.6×10^6 M−1s−1, respectively), while effects on fibrinogen clotting and susceptibility to hirudin inhibition varied profoundly among mutants. The authors interpret these findings in the context of known thrombin interactions with hirudin, thrombomodulin, rhodniin, and heparin cofactor II, concluding that ABE-I residues contribute via both general electrostatic steering and ligand-specific contacts. This is relevant to ASH's domain as it deepens mechanistic understanding of how hirudin binds thrombin's exosite I. However, the study focuses on thrombin molecular biology using mutagenesis; hirudin is discussed as a comparator ligand rather than being the primary subject of investigation.
Citation
The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1
Myles T et al. · European journal of biochemistry, 2001
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