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A factor XIa-activatable hirudin-albumin fusion protein reduces thrombosis in mice without promoting blood loss

Authors
  • Sheffield, William P.1, 2
  • Eltringham-Smith, Louise J.1
  • Bhakta, Varsha2
  • 1 McMaster University, Department of Pathology and Molecular Medicine, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada , Hamilton (Canada)
  • 2 Canadian Blood Services, Centre for Innovation, Hamilton, ON, Canada , Hamilton (Canada)
Type
Published Article
Journal
BMC Biotechnology
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Apr 05, 2018
Volume
18
Issue
1
Identifiers
DOI: 10.1186/s12896-018-0431-4
Source
Springer Nature
Keywords
License
Green

Abstract

BackgroundHirudin is a potent thrombin inhibitor but its antithrombotic properties are offset by bleeding side-effects. Because hirudin’s N-terminus must engage thrombin’s active site for effective inhibition, fusing a cleavable peptide at this site may improve hirudin’s risk/benefit ratio as a therapeutic agent. Previously we engineered a plasmin cleavage site (C) between human serum albumin (HSA) and hirudin variant 3 (HV3) in fusion protein HSACHV3. Because coagulation factor XI (FXI) is more involved in thrombosis than hemostasis, we hypothesized that making HV3 activity FXIa-dependent would also improve HV3’s potential therapeutic profile. We combined albumin fusion for half-life extension of hirudin with positioning of an FXIa cleavage site N-terminal to HV3, and assessed in vitro and in vivo properties of this novel protein.ResultsFXIa cleavage site EPR was employed. Fusion protein EPR-HV3HSA but not HSAEPR-HV3 was activated by FXIa in vitro. FVIIa, FXa, FXIIa, or plasmin failed to activate EPR-HV3HSA. FXIa-cleavable EPR-HV3HSA reduced the time to occlusion of ferric chloride-treated murine arteries and reduced fibrin deposition in murine endotoxemia; noncleavable mycHV3HSA was without effect. EPR-HV3HSA elicited less blood loss than constitutively active HV3HSA in murine liver laceration or tail transection but extended bleeding time to the same extent. EPR-HV3HSA was partially activated in citrated human or murine plasma to a greater extent than HSACHV3.ConclusionsReleasing the N-terminal block to HV3 activity using FXIa was an effective way to limit hirudin’s bleeding side-effects, but plasma instability of the exposed EPR blocking peptide rendered it less useful than previously described plasmin-activatable HSACHV3.

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