Noncanonical Proteolytic Activation of Human Prothrombin by Subtilisin from Bacillus subtilis may Shift the Procoagulant-Anticoagulant Equilibrium Toward Thrombosis [Protein Structure and Folding]

ABSTRACT Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)−mediated conversion of the prothrombin (ProT) zymogen to active αthrombin (αT). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg271, generating the inactive precursor prethrombin−2 (Pre2), which is further attacked at Arg320−Ile321 to yield mature αT. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nM−2 μM), a serine protease secreted by the nonpathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active σPre2, a nicked Pre2 derivative with a single cleaved Ala470−Asn471 bond. Notably, we found that this noncanonical cleavage at Ala470−Asn471 is instrumental for the onset of catalysis in σPre2, which was however reduced about 100−200 fold compared with αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by nonvirulent bacteria such as B. subtilis, can shift the delicate procoagulant−anticoagulant equilibrium toward thrombosis.

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