Bifunctional fusion protein targeting both FXIIa and FXIa displays potent anticoagulation effects

Abstract

Aims: Anticoagulation in disease treatment has been wildly studied in recent years. The intrinsic coagulation pathway is attracting attention of research community due to its low bleeding risk, and inhibitors against intrinsic coagulation factor XIIa (FXIIa) or XIa (FXIa) have been extensively studied. However, studies to develop anticoagulant inhibitors simultaneous targeting FXIIa and FXIa have not been reported. Our study aimed to evaluate the anticoagulation effect of the dual targeting of FXIIa and FXIa.

Main methods: A fusion protein Infestin-PN2KPI (IP) was designed by linking FXIIa inhibitor Infesin4 and FXIa inhibitor PN2KPI through a rigid linker, and was cloned, expressed and characterized. The binding of IP to FXIIa and FXIa was verified by SPR, and inhibitory ability of IP against FXIIa and FXIa was verified by chromogenic substrate method. And then, the anticoagulation and antithrombotic functions of IP were extensively evaluated by aPTT assay, FeCl₃-induced carotid artery thrombosis model and transient occlusion of the middle cerebral artery model.

Key findings: IP significantly prolonged aPTT, inhibited thrombosis and prevented stroke at a dose of at least 1/2 lower than the effective dose of its component Infestin4 or PN2KPI, and did not cause bleed risk.

Significance: The bifunctional fusion protein IP showed good anticoagulation effects, and simultaneous targeting FXIIa and FXIa is a promising strategy for anticoagulation drug development.

Keywords: Anticoagulation; Antithrombic; FXIIa; FXIa; Fusion protein.