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

William P. Sheffield,Varsha Bhakta,Louise J. Eltringham-Smith

doi:10.1186/s12896-018-0431-4

William P. Sheffield, Varsha Bhakta + Show 1 more

Open Access

https://doi.org/10.1186/s12896-018-0431-4

Copy DOI

Journal: BMC biotechnology	Publication Date: Apr 5, 2018
Citations: 7	License type: open-access

Affiliation: McMaster University, Canadian Blood Services

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.

Highlights

Hirudin is a potent thrombin inhibitor but its antithrombotic properties are offset by bleeding side-effects
Immunoblotting revealed that all P. pastoris-derived fusion proteins reacted with antiHSA (Fig. 2b) and anti-hexahistidine monospecific polyclonal antibodies (Fig. 2c), while only mycEPRHV3HSA and mycHV3HSA reacted with anti-myc monoclonal antibodies (Fig. 2d)
Our ability to activate EPRHV3HSA was entirely consistent with the findings of Zhang et al, who reported that hirudin variant 2 (HV2) activity was liberated by FXIa cleavage of recombinant protein EH, in which EPR was positioned on the N-terminus of HV2 [15]

Summary

Introduction

Hirudin is a potent thrombin inhibitor but its antithrombotic properties are offset by bleeding side-effects. In the biotechnological production of recombinant hirudin, it was noted that much of its activity was lost if its N-terminus was blocked, even by small peptides [10, 11] This observation was supported and rationalized by the co-crystallization of hirudin with thrombin, which showed the N-terminus of the small protein inserted into the active site canyon of the enzyme [12]. These findings prompted efforts to engineer a protease-activated “switch” into hirudin, to create a specific way of activating the thrombin inhibitor. Zhang et al capped the N-terminus of hirudin with small peptides cleavable by thrombin, FXa, or FXIa [15]

Methods

Results

Discussion

Conclusion