A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo
Research article published in Thromb Haemost (2009)
Abstract
The leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin's rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
HSACHV3, a plasmin-activatable HSA-HV3 hirudin chimera, shows slow clearance, plasmin-triggered thrombin inhibition, and lacks bleeding propensity in mouse models.
Why This Matters for Hirudotherapy
This study engineered a chimeric protein (HSACHV3) by fusing human serum albumin to the N-terminus of leech-derived hirudin variant 3 (HV3) with an intervening plasmin cleavage site, creating a thrombin inhibitor that is latent until activated by plasmin at sites of ongoing clot lysis. In vitro, HSACHV3 showed no thrombin inhibitory activity without plasmin but released active HV3 in a time- and concentration-dependent manner when plasmin was present, and assisted clot lysis in a tPA-dependent fashion. In mice, HSACHV3 cleared far more slowly than unfused HV3 and—unlike HV3 at equivalent doses—did not increase bleeding in a tail transection model. This work is highly relevant to ASH's domain because it directly tackles two major clinical limitations of recombinant hirudin: hemorrhagic risk and rapid clearance. However, all findings are preclinical (in vitro and mouse models), and no human efficacy or safety data are presented.
Citation
A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo.
Sheffield WP et al. · Thromb Haemost, 2009
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