[30] Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin

Olson, Steven T.; Björk, Ingemar; Shore, Joseph D.

doi:10.1016/0076-6879(93)22033-c

Cited by 277 publications

(507 citation statements)

References 109 publications

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“…Heparinactivated Antithrombin-Heparin enhances the rate of FXa inhibition by antithrombin by ϳ1000-fold (28). Structural data suggest that residues of the reactive site loop of antithrombin have a non-optimal conformation for interacting with FXa, but that binding of heparin to antithrombin alters this conformation and/or frees the loop, allowing it to fit into the catalytic groove of the target (48).…”

Section: Role Of Loop 34 -40 Of Fxa In the Interaction Withsupporting

confidence: 88%

“…The antithrombin⅐polysaccharide concentration was estimated using Equation 3 in which antithrombin⅐polysaccharide was substituted for FVa⅐FXa, and the concentration of heparin was substituted for FVa 0 and that of antithrombin for FXa 0 . The K D of the antithrombin⅐polysaccharide complex was determined by intrinsic fluorescence measurement as described by Olson et al (28).…”

Section: Methodsmentioning

confidence: 99%

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The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

Bianchini

Pike

Bonniec

2004

Journal of Biological Chemistry

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Coagulation factor X (FX) 1 is a vitamin K-dependent zymogen activated into FXa by cleavage at a single bond (Arg 15 -Ile 16 in the chymotrypsin numbering system 2 ) hydrolyzed by either of two complexes: FVIIa bound to tissue factor (TF), which triggers coagulation after injury, or FIXa associated with FVIIIa, which amplifies thrombin production after initiation of coagulation. FXa is the only endogenous prothrombin activator, but substantial thrombin production occurs only within the prothrombinase complex, where FXa binds to FVa in the presence of calcium on an appropriate phospholipid surface. Two inhibitors control FXa activity, viz. the tissue factor pathway inhibitor (TFPI) and antithrombin.Extended binding sites (exosites) play a crucial role in virtually any substrate, cofactor, or inhibitor recognition within the coagulation cascade. Perhaps best characterized is the case of thrombin taking advantage of two exosites for extended interactions in numerous functions (1). Exosite-1 is formed by a set of basic residues comprising loops 34 -40 and 70 -80, which neighbor each other in the folded structure (2); it interacts with fibrinogen, thrombomodulin, platelet activator receptor-1, FV, FVa, heparin cofactor II, and hirudin. Exosite-2, comprising loop 91-102 and helices 165-173 and 233-245 (3), is also formed by a set of basic residues that interact with heparin, with the chondroitin sulfate of thrombomodulin, and within prothrombin with kringle-2 (4). In FVII, the region corresponding to exosite-1 of thrombin is critical for FX activation (5, 6); in FIXa, this region is also important for FX activation in the absence of FVIII and for its inhibition by antithrombin in the absence of heparin (7). In contrast to thrombin, FVIIa, FIXa, and FXa share a negatively charged patch within segment 70 -80, whereas FXa is unique in that both loops 34 -40 and 70 -80 are negatively charged. Thus, the region of FXa corresponding to exosite-1 of thrombin forms a negative cluster, raising the question as to whether it could constitute a functional exosite. FXa has a unique macromolecule substrate, but exosites may also participate in its inhibition and/or in the activation of its zymogen. In fact, a number of studies have established that exosite(s) must be critical in several FXa functions. Electrostatic interactions involving loop 34 -40 of FXa appear to play a significant role in binding to the second Kunitz domain of TFPI (8,9). FXa also appears to interact with a complementary surface on pentasaccharide-activated antithrombin (10), and the region of FXa topologically equivalent to exosite-2 of thrombin seems to be involved in the binding of heparin and FVa (11). Above all, prothrombin recognition by prothrombinase undoubtedly involves one or more exosites on .In a previous study (15), we reported that the catalytic groove of FXa is minimally selective with unconstrained peptides and that FVa has little effect, if any, on this selectivity. These two observations therefore also favor the hypothesis that FXa uses seconda...

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Section: Role Of Loop 34 -40 Of Fxa In the Interaction Withsupporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

Bianchini

Pike

Bonniec

2004

Journal of Biological Chemistry

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“…Kinetics of Trypsin or HNE Inhibition by DCI or Chloromethyl Ketones-The kinetics of enzyme inhibition were measured under pseudo first-order conditions either by discontinuous or continuous assay methods (33). For the discontinuous assay method, reactions contained 10 nM enzyme and either 125-250 M DCI, 100 -200 nM Phe-Phe-Arg chloromethyl ketone (Calbiochem), or 5 M methoxysuccinyl-Ala-Ala-ProVal chloromethyl ketone (Bachem, King of Prussia, PA) in 0.1 ml.…”

Section: Methodsmentioning

confidence: 99%

“…The exponential decrease in substrate hydrolysis rate was monitored continuously for up to 5 min to an end point rate (Ͻ2% of the initial rate) with Ͻ5% consumption of substrate. Progress curves were fit by nonlinear regression to an exponential plus linear term (33,34) to obtain k obs . k obs was corrected for substrate competition by multiplying by the factor, 1 ϩ [S] 0 /K m , where [S] 0 is the substrate concentration.…”

Section: Methodsmentioning

confidence: 99%

“…A 50-l aliquot containing 47-140 nM DCI HNE or 5-16 nM DCI trypsin was then added to 0.95 ml of 200 M substrate in buffer containing 4 -5% Me 2 SO, and the accelerating rate of substrate hydrolysis was monitored continuously at 405 nm for 15 min at 37°C (Յ5% substrate hydrolysis). Data were fit by nonlinear regression by the parabolic equation (33),…”

Section: Methodsmentioning

confidence: 99%

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Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

Olson

Swanson²,

Patston

et al. 1997

Journal of Biological Chemistry

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Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thøgersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859 -14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and ␤-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, ␣ 1 -proteinase inhibitor (␣ 1 PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a ratelimiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the ␣ 1 PI reactive center P1-P1 bond. Quantitation of kinetic constants for DCI and ␣ 1 PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by ␣ 1 PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

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Overview of Hemostasis

Mann¹,

Ziedins²

2005

Textbook of Hemophilia

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[30] Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin

Cited by 277 publications

References 109 publications

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

Overview of Hemostasis

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