The action of Factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions

Lottenberg, Richard; Hall, Julie; Pautler, Ellen; Zupan, Andrew; Christensen, Ulla; Jackson, Craig M.

doi:10.1016/0167-4838(86)90032-4

Cited by 66 publications

(56 citation statements)

References 25 publications

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“…An obvious explanation for this finding has not been forthcoming since identical P 1 -P 4 residues precede both cleavage sites in prothrombin (36). Assuming that Scheme II provides an adequate description of the binding steps in substrate recognition, the present results indicate that the rate constant for catalysis is essentially the same for the two cleavage reactions and is comparable to values observed for the cleavage of peptidyl substrates bearing the same P 1 -P 4 sequence encountered in the protein substrate (57).…”

Section: Discussionmentioning

confidence: 51%

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Boskovic

Krishnaswamy²

2000

Journal of Biological Chemistry

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The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg 323 -Ile 324 , followed by cleavage at Arg 274 -Thr 275 . We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIa⌬F1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIa⌬F1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K m . This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIa⌬F1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIa⌬F1 to the enzyme. Specific recognition of mIIa⌬F1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.Prothrombinase is an archetypal enzyme complex of blood coagulation (2). The enzyme complex assembles through well characterized, reversible, protein-protein and protein-membrane interactions between the serine protease, factor Xa, the cofactor, factor Va, and membranes in the presence of calcium ions (2-4). The resulting complex catalyzes the conversion of prothrombin to thrombin at a greatly enhanced rate, compared with the reaction rate catalyzed by factor Xa alone (2).Prothrombin is activated by proteolytic cleavage at two sites, Arg 274 -Thr 275 and Arg 323 -Ile 324 , which yields the fragment 1.2 activation peptide and thrombin 1 (5, 6). The reaction catalyzed by prothrombinase proceeds almost exclusively via the initial cleavage at Arg 323 -Ile 324 , yielding meizothrombin as an intermediate, followed by cleavage at Arg 274 -Thr 275 to yield the final products of the reaction (7,8). Single turnover kinetic studies indicate that the overall process is likely the sum of two consecutive enzyme-catalyzed reactions (8). Consequently, steady state...

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Section: Discussionmentioning

confidence: 51%

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Boskovic

Krishnaswamy²

2000

Journal of Biological Chemistry

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“…In the RE-ALIGN study, mean dabigatran levels were determined using the thrombin-based Hemoclot coagulation assay and were similar [37] to those reported by others [11] and in our patients. Results for the two chromogenic substrates with different selectivity and specificity toward thrombin support this conclusion because a high congruency of data was found [38,39]. In contrast, another direct synthetic inhibitor, argatroban, could not be detected by chromogenic substrate assays with a sensitivity that was high enough for clinical samples [40].…”

Section: Discussionmentioning

confidence: 72%

Determination of dabigatran in plasma, serum, and urine samples: comparison of six methods

Du¹,

Weiß²,

Christina³

et al. 2015

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background: Assessing the anticoagulant effect of dabigatran may be useful in certain clinical settings. When plasma sampling is not available, serum or urine samples may provide another option for dabigatran determinations. Methods: Dabigatran was assessed in patients on treatment under real-life conditions in plasma samples by four clotting time-based assays and in plasma, serum, and urine samples by two chromogenic substrate methods. Results: The concentrations of dabigatran in patients' plasma samples were not different for the Hemoclot test (106.8 ± 89.4 ng/mL) and the ecarin clotting time (ECT, 109.5 ± 74.5 ng/mL, p = 0.58). Activated partial thromboplastin time and prothrombinase-induced clotting time showed low correlations with the other assays. Chromogenic assays measured similar concentrations as Hemoclot and ECT. For both chromogenic assays, the concentrations of dabigatran were about 70% lower in serum than in plasma samples (p < 0.0001). The intra-class coefficient (ICC, Bland-Altman analysis) was strong comparing ECT, Hemoclot thrombin inhibitor (HTI) assay, and the two chromogenic assays (r = 0.889-0.737). The ICC was low for comparisons of the chromogenic assays of serum vs. plasma values (ICC, 0.15 and 0.66). The ICC for the determination of

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“…genic substrates (37)(38)(39), and indeed, optimization of the activation domain of prethrombin-2 for thrombin cleavage has recently involved mutation of Gly-14m (40). Remarkably, the G14mP single mutant of prethrombin-2 autoactivates as rapidly as the DE double mutant and faster than the EDE triple mutant (Fig.…”

Section: Resultsmentioning

confidence: 99%

Autoactivation of Thrombin Precursors

Pozzi

Chen

Zapata

et al. 2013

Journal of Biological Chemistry

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Background: Thrombin is generated from zymogen precursors with the assistance of cofactors. Results: Thrombin precursors prethrombin-2 and prothrombin are capable of catalytic activity and autoactivate. Conclusion: Conformational selection regulates activity in the mature protease and has the potential to unleash autoactivation in the zymogen. Significance: The paradigm of the inactive zymogen to active protease conversion needs revision to account for conformational selection.

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The action of Factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions

Cited by 66 publications

References 25 publications

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Determination of dabigatran in plasma, serum, and urine samples: comparison of six methods

Autoactivation of Thrombin Precursors

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