Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Filippis, Vincenzo De; Russo, Ilaria; Vindigni, Alessandro; Di, Enrico; Salmaso, Stefano; Fontana, Angelo

doi:10.1110/ps.8.10.2213

Cited by 22 publications

(27 citation statements)

References 32 publications

(23 reference statements)

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“…An identical amount (100 pmol) of purified ␣T A-and B-chain or Pre2 NT and CT fragments were loaded on a Procise sequencer (Applied Biosystems). Prior to analysis, these chains/ fragments were subjected to disulfide bond reduction and carboxamidomethylation with dithiothreitol and iodoacetamide (75) and subsequent purification by RP-HPLC. As a reference, sequence analyses were performed on the corresponding species derived from ␣T and Pre2 samples that were not treated with KCNO.…”

Section: N-terminal Carbamylationmentioning

confidence: 99%

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

Pontarollo

Acquasaliente

Peterle

et al. 2017

Journal of Biological Chemistry

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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). ...

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Section: N-terminal Carbamylationmentioning

confidence: 99%

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

Pontarollo

Acquasaliente

Peterle

et al. 2017

Journal of Biological Chemistry

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“…The N-terminal domain 1-47 of hirudin ( Figure 5A) extensively interacts with thrombin recognition sites ( Figure 5B) and, like intact hirudin [5,41] and fibrinogen [4,5], binds preferentially to the fast form of the enzyme. Therefore, it may serve as a reliable structural probe for the transition of thrombin from the slow into the fast form.…”

Section: Structural Mapping Of Thrombin Recognition Sites In the Fastmentioning

confidence: 99%

Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

Filippis

Dea

Lucatello

et al. 2005

Biochemical Journal

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In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by approximately 2 kcal/mol (1 cal identical with 4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the 'fast' form. Finally, we have used hirudin fragment 1-47 as a molecular probe of the conformation of thrombin recognition sites in the fast and 'slow' form. From the effects of amino acid substitutions on the affinity of fragment 1-47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.

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“…Natural and recombinant desulphated hirudin have similar biological and anticoagulant activities (11,12). Natural hirudin is a single-chain, carbohydrate-free polypeptide composed of 65 amino acids (molecular weight: 7 kDa).…”

Section: Introductionmentioning

confidence: 99%

The Use of Hirudin as Universal Anticoagulant in Haematology, Clinical Chemistry and Blood Grouping

Menssen¹,

Melber²,

Brandt³

et al. 2001

Clinical Chemistry and Laboratory Medicine

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To evaluate the serum urate levels in AIDS patients with infections of the central nervous system (CNS), 46 patients who had at least two measurements of urate were included. A maximum of four measurements per patient were considered: prior to the CNS involvement (U-PRIOR), at the time of CNS involvement (U-CNS), after treatment for the CNS infection (U-AFTER), and the last measurement before death (U-LAST). Serum U-CNS levels were significantly lower than U-PRIOR values (p=0.038). U-AFTER levels were higher than U-CNS in the patients who improved (p=0.25), and lower in the patients who did not improve (p=0.026). There were no significant differences among the four diagnostic groups in U-CNS measurement (p=0.29) but they were found in U-AFTER determinations (p=0.018), probably as a result of the different response to treatment. Hypouricemia seemed to be associated with lower survival periods. We conclude that hypouricemia is common in AIDS patients with CNS infections, probably as a result of increased renal losses of urate, and that it may have prognostic significance. CNS infections are associated with significant decreases in serum urate levels in comparison with previous values. The urate concentrations seem to increase after successful treatment of the CNS infections, whereas they decrease further in patients who do not improve.

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Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Cited by 22 publications

References 32 publications

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

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

Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

The Use of Hirudin as Universal Anticoagulant in Haematology, Clinical Chemistry and Blood Grouping

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