Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant–anticoagulant equilibrium toward thrombosis

Abstract

Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active ␣-thrombin (␣T). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320 -Ile-321 to yield mature ␣T. 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 to 2 M), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active Pre2, a nicked Pre2 derivative with a single cleaved Ala-470 -Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470 -Asn-471 is instrumental for the onset of catalysis in Pre2, which was, however, reduced about 100 -200-fold compared with ␣T. Of note, Pre2 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 non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.Blood coagulation is a finely regulated physiological process that culminates with the factor Xa-mediated conversion of the prothrombin (ProT) 3 zymogen to the active ␣-thrombin (␣T) enzyme, which in turn is responsible for the generation of insoluble fibrin and activation of platelets via the GpIb␣-PAR1 pathway (1, 2). ProT (ϳ72 kDa) is a vitamin K-dependent glycoprotein produced in the liver and circulating at a relatively high plasma concentration (0.1 mg/ml) (3). The domain architecture of ProT includes a Gla domain (residues 1-46), a kringle-1 (residues 65-143) and a kringle-2 (residues 170 -248) domain, and a chymotrypsin-like protease domain (residues 285-579) connected by three intervening linker regions (Lnk-1, -2, and -3) (4). Isolated factor Xa (FXa) has low intrinsic ProTconverting activity, but when it is assembled in the presence of Ca 2ϩ with cofactor Va in the prothrombinase complex on the platelet surface, its ability to activate ProT is increased by about 5 orders of magnitude (5). FXa cleaves ProT in a concerted manner at two sites, i.e. Arg-271 and Arg-320, but the order of peptide bond cleavage is highly context-dependent. On the platelet surface, FXa first cleaves ProT at Arg-271 generating the inactive precursor prethrombin-2 (Pre2), which is attacked by FXa at Arg-320 to generate the active ␣T species, formed by the polypeptide chains Thr-272-Arg-320 and Ile-321-Glu-579 (6). ...

Interplay between conformational selection and zymogen activation

“…Independent support to the role of conformational selection revealed by rapid kinetics comes from limited proteolysis experiments. Interest in this aspect of zymogen function comes from the recent observation that prothrombin is proteolytic attacked by subtilisin at several residues to produce prethrombin-1, prethrombin-2 and eventually a derivative of prethrombin-2 cleaved at A470 in the flexible autolysis loop that is activated independent of the Huber-Bode mechanism 32 . The rate of proteolytic digestion of prothrombin by subtilisin is significantly enhanced in the presence of the active site inhibitor argatroban (Fig.…”

Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation

“…Epithelial PAR signaling is of great relevance in IBD and was previously reported for other bacterial proteases including Enterococcus faecalis , Porphyromonas gingivalis, and Clostridium difficile 133‐135 . Besides pathogenic bacteria, proteases released by even non‐virulent bacteria, such as Bacillus subtilis, was shown to activate the human prothrombin and shift the procoagulant‐anticoagulant equilibrium toward thrombosis, known as most common events in patients with IBD 136,137 . This picture is far from complete since culprit proteolytic bacteria remain largely unknown as well as their mechanisms of action.…”

Structure of Coagulation Factor II: Molecular Mechanism of Thrombin Generation and Development of Next-Generation Anticoagulants

“…In the closed form, kringle-1 sits on top of the catalytic pocket, therefore, blocking access of substrates to the active site, as well as hiding portions of the flexible autolysis loop. Given that prothrombin undergoes autoactivation when histones are dumped in the circulation upon cellular damage ( 24 , 36 ), and that the autolysis loop is targeted by bacterial proteases that generate thrombin bypassing the canonical activation pathway ( 37 ), the closed conformation of prothrombin may represent an autoinhibited, proteolytically resistant form of the zymogen in the circulation with improved plasma half-life. This protection mechanism may be shared with other plasma proteins such as factor XII, which has been recently shown to have single-chain catalytic activity ( 26 ).…”