A human-cell recombinant prethrombin-2 platform producing thrombin with enhanced fibrinogen processing for in vitro...

Thrombin is a central mediator of haemostasis, immune responses, and inflammation. It arises from prothrombin upon prothrombinase cleavage at R³²⁰-I³²¹, which triggers the allosteric maturation typical of chymotrypsin-like serine peptidases. Further processing yields the physiologically dominant α-thrombin (αT), whose competent active site centres on catalytic S⁵²⁵. Despite widespread biomedical and biotechnological use, plasma-derived αT is heterogeneous and risks pathogen or prion transmission, motivating recombinant alternatives. These have largely relied on suboptimal bacterial or non-human eukaryotic systems, with human-cell efforts focused mainly on prothrombin or engineered variants. Here, we report the recombinant production in Expi293F cells of prethrombin-2 (PE2), a zymogen retaining 13 residues upstream of αT, alongside its catalytically-impaired S⁵²⁵A counterpart (PE2-S⁵²⁵A). Controlled in vitro maturation was achieved with a matched recombinant system for ecarin, a snake-venom metallopeptidase cleaving R³²⁰-I³²¹, which yielded active PE2a and inactive PE2a-S⁵²⁵A. PE2a exhibited thermal stability, cleavage activity against the chromogenic substrate S-2238, and performance in thrombin-time assays comparable to those of commercial human αT. By contrast, it cleaved the physiological substrate fibrinogen, including its fluorescein-labelled form, more efficiently. Fresh PE2a slowly underwent autolysis at R²⁸⁴-T²⁸⁵, yielding αT after one week. The direct thrombin inhibitors argatroban and dabigatran further enhanced thermal stability while efficiently blocking substrate cleavage. This fully recombinant workflow provides a practical platform for basic haematological research, structural biology, and inhibitor screening in vitro.