The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand

Abstract

BackgroundHeparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli.ResultsImmobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 μM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1.ConclusionsAssuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.

Fusion of the C-terminal triskaidecapeptide of hirudin variant 3 to alpha1-proteinase inhibitor M358R increases the serpin-mediated rate of thrombin inhibition

“…A small recombinant protein, 84 amino acids in size, comprising residues 1-75 of HCII with an N-terminal nonapeptide tag MSGH 6 (designated HCII 1-75), was expressed in a pBAD-based plasmid and purified from sonicated cell lysates by nickel-chelate affinity chromatography as described [ 36 ].…”

“…Human citrated plasma was combined in a 1:3:5 volume ratio of plasma: veronal buffer: Thrombin 10 calcified thrombin reagent, and the time to clot was determined. The veronal buffer (sodium acetate trihydrate 7.14 mM/ sodium diethyl barbiturate 7.4 mM/ NaCl 0.131 M pH7.4) was employed with or without supplementation with purified recombinant serpins or synthetic peptides, as described [ 36 ].…”

“…A previously described assay was employed in order to characterize the relative affinity of recombinant serpins and peptides for either α-thrombin or α-thrombin rendered inactive at its active site by incubation with FPR-chloromethylketone [ 36 ]. Briefly, purified HCII 1-75 was immobilized on microtiter plates and purified human α-thrombin was incubated with or without competitor peptides or proteins.…”

“…The acidic extension of HCII accelerates thrombin inhibition by binding to thrombin exosite 1, a cluster of charged residues involved in binding thrombin co-factors such as thrombomodulin and substrates such as fibrinogen and coagulation factor V [ 30 ]. This deduction is supported by: a partially resolved X-ray crystal structure of the HCII-S195A thrombin structure [ 31 ]; reduced rates of inhibition of HCII and HCII fusion proteins on exosite-1-disrupted forms of thrombin [ 32 - 35 ]; and direct binding studies of either HCII 1-75 or its smaller derivatives [ 36 , 37 ]. The leech anticoagulant protein hirudin is the most potent known polypeptide inhibitor of thrombin [ 38 ], and owes some of its high affinity for thrombin to the binding of its 11-13 C-terminal residues, depending on the isoform [ 39 ], to thrombin exosite 1.…”

“…The leech anticoagulant protein hirudin is the most potent known polypeptide inhibitor of thrombin [ 38 ], and owes some of its high affinity for thrombin to the binding of its 11-13 C-terminal residues, depending on the isoform [ 39 ], to thrombin exosite 1. We [ 36 ] and others [ 37 ] have demonstrated that hirudin C-terminal peptides bind more tightly to thrombin than HCII 1-75 or its derivatives. Accordingly, in the current study we tested the primary hypothesis that fusion of the C-terminal 13 amino acids of hirudin variant 3 (HV3), the most potent hirudin isoform [ 39 ], to API M358R, would increase the rate of thrombin inhibition of API M358R by conferring on this mutant serpin the ability to bind thrombin exosite 1.…”