Heparin-catalyzed inhibitor/protease reactions: kinetic evidence for a common mechanism of action of heparin.

Griffith, Michael

doi:10.1073/pnas.80.18.5460

Cited by 59 publications

(20 citation statements)

References 18 publications

(12 reference statements)

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“…15 Simultaneous binding of serine protease inhibitors (serpins) and serine proteases to heparin results in inhibition. 31, 32 Considering such alternative hypotheses and elucidating the mechanism of GAG activation of proMMP-7 is the purpose of this work.…”

Section: Introductionmentioning

confidence: 99%

Heparinoids Activate a Protease, Secreted by Mucosa and Tumors, via Tethering Supplemented by Allostery

Fulcher

Gari²,

Frey³

et al. 2014

ACS Chem. Biol.

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Activation by glycosaminoglycans (GAGs) is an emerging trend among extracellular proteases important in disease. ProMMP-7, the zymogen of a matrix metalloproteinase secreted by mucosal epithelial and tumor cells, is activated at their surfaces by sulfated GAGs, but how? ProMMP-7 is activated in trans by representative heparin oligosaccharides in a length-dependent manner, with a large jump in activation at lengths of 16 monosaccharides. Imaging by atomic force microscopy visualized small complexes of proMMP-7 molecules linked by 8-mer lengths of heparinoids and extended assembles formed with 16-mer lengths of heparin. Complexes of proMMP-7 with polydisperse heparin or heparan sulfate were more diverse. Heparinoids evidently accelerate activation by tethering multiple proMMP-7 molecules together for proteolytic attack among neighbors. Removal of either the prodomain or C-terminal peptide sequence of KRSNSRKK from MMP-7 prevents formation of the long arrays induced by heparin 16-mers or heparan sulfate. The role of the C-terminus in activation assays suggests it contributes to remote, allosteric binding of GAGs. Enhancement of proteolytic velocity of MMP-by GAGs indicates them to be effectors of V-type allostery. GAGs from proteoglycans appear to assemble proMMP-7 molecules for activation, an event preceding its tumorigenic or anti-bacterial proteolytic activities at cell surfaces.

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Section: Introductionmentioning

confidence: 99%

Heparinoids Activate a Protease, Secreted by Mucosa and Tumors, via Tethering Supplemented by Allostery

Fulcher

Gari²,

Frey³

et al. 2014

ACS Chem. Biol.

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“…The accelerating effect of different GAGs on α‐thrombin inhibition by HCII includes a complex series of processes, the relative importance of which may depend on the nature of the activating polyanion. Heparin and dermatan sulfate have been suggested to act as a template for surface approximation of enzyme and inhibitor [15,16]. Other work suggests that GAGs may liberate the acidic N‐terminal domain of HCII from intramolecular interactions by displacement, providing an exosite for binding to α‐thrombin [9,10,17], eventually in combination with a bridging mechanism [1,17,18].…”

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confidence: 99%

Reformable intramolecular cross‐linking of the N‐terminal domain of heparin cofactor II

Brinkmeyer

Eckert

Ragg

2004

European Journal of Biochemistry

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The crystal structure of a heparin cofactor II (HCII)-thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we demonstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition of a-chymotrypsin, indicating that the reactive site loop sequences of the engineered inhibitor variants, required for interaction with one of the HCII target enzymes, are normally accessible. In contrast, intramolecular tethering of the N-terminal extension results in a drastic decrease of a-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards a-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and a-thrombin.Keywords: a-thrombin; dermatan sulfate; heparin cofactor II; heparin; serpin(s).Heparin cofactor II (HCII), a member of the serpin family, is an efficient inhibitor of a-thrombin in the presence of a variety of polyanions, including glycosaminoglycan (GAGs) such as heparin or dermatan sulfate [1]. In the absence of these compounds, the rate of a-thrombin inhibition is lowered by several orders of magnitude. HCII also inhibits a-chymotrypsin [2] and cathepsin G [3]; the reaction rates, however, are only moderately affected by GAGs. In recent years, substantial evidence has been accumulated on the molecular basis underlying the inhibition of serine proteinases by serpins [4,5]. Key features of the mechanism include the presentation of the inhibitor's reactive site loop (RSL) to a target enzyme, the initial cleavage of the scissile bond within the RSL, and formation of a covalent acyl ester intermediate between the catalytic serine of the enzyme and the carboxyl group of the P1 residue of the RSL. In the inhibitory path of the branched pathway mechanism of serpins, the RSL is inserted into b-sheet A with concomitant translocation of the attached proteinase to the opposite pole of the inhibitor [6,7].For most serpins, the specificity of the inhibitor-enzyme reaction is primarily determined by residues within the RSL, with a dominant importance of residues flanking the scissile bond. However, exosite interactions can also play an important role, as recently demonstrated for the heparininduced acceleration of inhibition of factors Xa and IXa by antithrombin [8]. Exosite contacts have also been implicated in the GAG-enhanced a-thrombin inhibition by HCII [9][10][11], and the crystal st...

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“…[1][2][3] The mechanism and the extent of this acceleration have been thoroughly investigated for antithrombin inhibition of factor Xa and thrombin. [4][5][6][7][8][9][10][11][12][13][14][15] It is not, however, known whether heparin is effective in catalyzing the inhibition of factor Xa when the proteinase binds to factor Va on negatively charged membrane surfaces in the presence of calcium (the prothrombinase complex) to activate prothrombin to thrombin. [16][17][18][19][20][21][22][23][24] Results of several previous studies suggest that the assembly of factor Xa into the prothrombinase complex is accompanied by the protection of factor Xa from inhibition by the antithrombin-heparin complex.…”

Section: Introductionmentioning

confidence: 99%

Prothrombin protects factor Xa in the prothrombinase complex from inhibition by the heparin-antithrombin complex

Rezaie¹

2001

Blood

100

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Heparin is a commonly used anticoagulant drug. It functions primarily by accelerating the antithrombin inhibition of coagulation proteinases, among which factor Xa and thrombin are believed to be the most important targets. There are conflicting results as to whether anticoagulant heparins can catalyze the antithrombin inhibition of factor Xa in the prothrombinase complex (factor Va, negatively charged membrane surfaces, and calcium ion), which is the physiologically relevant form of the proteinase responsible for the activation of prothrombin to thrombin during the blood coagulation process. In this study, a novel assay system was developed to compare the catalytic effect of different molecularweight heparins in the antithrombin inhibition of factor Xa, either in free form or assembled into the prothrombinase complex during the process of prothrombin activation. This assay takes advantage of the unique property of a recombinant mutant antithrombin, which, similar to the wild-type antithrombin, rapidly inhibits factor Xa, but not thrombin, in the presence of heparin. A direct prothrombinase inhibition assay, monitoring thrombin generation under near physiological concentrations of prothrombin and antithrombin in the presence of therapeutic doses of low-and high-molecular-weight heparins, indicates that factor Xa in the prothrombinase complex is protected from inhibition by antithrombin more than 1000 times, independent of the molecular size of heparin. IntroductionThe anticoagulant function of heparin is primarily derived from its ability to accelerate the inhibition of the blood coagulation proteinases by antithrombin. [1][2][3] The mechanism and the extent of this acceleration have been thoroughly investigated for antithrombin inhibition of factor Xa and thrombin. [4][5][6][7][8][9][10][11][12][13][14][15] It is not, however, known whether heparin is effective in catalyzing the inhibition of factor Xa when the proteinase binds to factor Va on negatively charged membrane surfaces in the presence of calcium (the prothrombinase complex) to activate prothrombin to thrombin. [16][17][18][19][20][21][22][23][24] Results of several previous studies suggest that the assembly of factor Xa into the prothrombinase complex is accompanied by the protection of factor Xa from inhibition by the antithrombin-heparin complex. [18][19][20][21] Conflicting reports exist about the extent to which factor Xa in the prothrombinase complex is protected from inhibition by antithrombin in the presence of different molecular weight heparins. These reports range from support for nearly complete protection 19,21 to less than 5-to 10-fold protection of factor Xa in prothrombinase from inhibition by antithrombin in the presence of high-or low-molecular-weight high-affinity heparins. [22][23][24] As expected, these results have generated conflicting hypotheses within the scientific community as to whether the therapeutic heparins are effective in catalyzing prothrombinase inhibition by antithrombin, and if so, whether the effect is dependent...

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Heparin-catalyzed inhibitor/protease reactions: kinetic evidence for a common mechanism of action of heparin.

Cited by 59 publications

References 18 publications

Heparinoids Activate a Protease, Secreted by Mucosa and Tumors, via Tethering Supplemented by Allostery

Heparinoids Activate a Protease, Secreted by Mucosa and Tumors, via Tethering Supplemented by Allostery

Reformable intramolecular cross‐linking of the N‐terminal domain of heparin cofactor II

Prothrombin protects factor Xa in the prothrombinase complex from inhibition by the heparin-antithrombin complex

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