Structure-function and refolding studies of the thrombin-specific inhibitor hirudin

We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Recombinant desulfo-hirudin HV-1 (10,000 antithrombin units/mg; Ki 0.2 pM) refolds rapidly at pH 10, behaves as a multimer that dissociates on thrombin binding, and inhibits via multiple noncovalent contacts; isolated fragments 42-65 and 51-65 also inhibited fibrinogen cleavage (IC50 4.9, 6.0 microM).

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