A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo

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.

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…With respect to the first hypothesis, and as predicted by Wu et al [24], KPI was rapidly removed from the murine circulation after intravenous injection. KPI clearance resembled that of other small proteins less than 80 amino acids in length such as hirudin [25, 35] or barbourin [36], which are characterized by rapid renal losses without reabsorption. Specifically with respect to recombinant hexahistidine-tagged hirudin variant 3 (HV3), we previously noted that ∼90% of the radiolabeled dose was lost from the murine circulation within one hour of injection, a profile highly similar to that we observed for KPI in the current study [25].…”

“…KPI clearance resembled that of other small proteins less than 80 amino acids in length such as hirudin [25, 35] or barbourin [36], which are characterized by rapid renal losses without reabsorption. Specifically with respect to recombinant hexahistidine-tagged hirudin variant 3 (HV3), we previously noted that ∼90% of the radiolabeled dose was lost from the murine circulation within one hour of injection, a profile highly similar to that we observed for KPI in the current study [25]. Fusion to albumin slowed both the rapid and slower phases of KPI’s biphasic clearance by 6.5- to 8.5-fold, providing a clear pharmacokinetic enhancement.…”

“…DNA sequences encoding KPI, appropriate for in-frame fusion to a human serum albumin (HSA) cDNA, were assembled using two sequential PCR steps. First, primers PrPKI-1 and ML-08-6225 were used to amplify plasmid pPICZ9ssHV3HSAH6 [25], yielding a 1243 bp product. Following gel purification, this DNA product was amplified again, substituting PrKPI-2 for PrPKI-1.…”

“…Following gel purification, this DNA product was amplified again, substituting PrKPI-2 for PrPKI-1. The second 1339 bp amplification product was restricted with XhoI and XbaI and inserted between these sites in pPICZ9ssHSAH 6 [25], yielding pPICZ9ssKPIHSAH 6 . This plasmid contained an open reading frame combining the 80 amino acid prepro-α factor secretory signal sequence (ss), the 57 amino acid KPI domain, a Gly 6 SerMet spacer, the 584 codons of mature HSA, and a C-terminal hexahistidine tag to facilitate purification.…”

“…This plasmid contained an open reading frame combining the 80 amino acid prepro-α factor secretory signal sequence (ss), the 57 amino acid KPI domain, a Gly 6 SerMet spacer, the 584 codons of mature HSA, and a C-terminal hexahistidine tag to facilitate purification. Plasmid pPICZ9ssKPIHSAH 6 was employed to transform Pichia pastoris strain X-33 to Zeocin resistance, transformed cell lines were cultured and induced with methanol, and secreted protein KPIHSA was purified from conditioned media using nickel-chelate affinity chromatography, as previously described [25]. …”