Structure-function relationships in heparin cofactor II: Spectral analysis of aromatic residues and absence of a role for sulfhydryl groups in thrombin inhibition

Church, Frank C.; Meade, J. B.; Pratt, Charlotte W.

doi:10.1016/0003-9861(87)90499-1

Cited by 24 publications

(14 citation statements)

References 37 publications

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“…It can be postulated that the carbohydrate content of RbHCII might be higher than that of human as it contain 5 N-glycosylation sites. It was reported that the antithrombin III (ATIII), a functional homologue of HCII, contains an essential disulfide bond for heparin-dependent thrombin inhibition [45]. However, similar to human HCII, no disulfide bond was predicted in RbHCII even though both HCIIs contain 3 Cys residues.…”

Section: Discussionmentioning

confidence: 99%

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

Umasuthan

Whang

Lee

et al. 2011

Fish & Shellfish Immunology

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Section: Discussionmentioning

confidence: 99%

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

Umasuthan

Whang

Lee

et al. 2011

Fish & Shellfish Immunology

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“…α-Thrombin, prepared as described [38], was ≥90% active as determined by active site titration [39]. Protein concentrations were determined by absorbance at 280 nm with the absorption coefficients and molecular weights ( M r ) of 1.83 (mg/ml) −1 cm −1 and 36,700, for thrombin [40]; 0.59 and 65,600 for plasma HCII [3,5]; and 0.73 and 54,960 for recombinant HCII [41,42]. Active HCII concentrations were determined by stoichiometric titration with thrombin in DS-accelerated reactions.…”

Section: Methodsmentioning

confidence: 99%

“…Thrombin is inactivated by circulating plasma heparin cofactor II (HCII),1 a member of the superfamily of serine proteinase inhibitors (serpins) [1–3]. Serpins possess a reactive center loop (RCL) that is cleaved by their target proteases, with formation of a stabilized, covalent serpin–enzyme complex in which the protease active site is distorted.…”

mentioning

confidence: 99%

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli

Sarilla

Habib

Tollefsen

et al. 2010

Analytical Biochemistry

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Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K D ) of 6 ± 1 and 7 ± 1 μM, respectively, ~6-fold tighter than plasma HCII, with K D 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with K D 4 ± 1 μM, was ~4-fold tighter than for plasma HCII, with K D 15 ± 4 μM. Recombinant HCII, lacking Nglycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn 169 N-glycan with the HCII heparin-binding site. KeywordsRecombinant heparin cofactor II; Serpin; Thrombin; Serine protease inactivation Thrombin, the central proteinase in the coagulation cascade, cleaves soluble fibrinogen to insoluble fibrin which polymerizes to form a blood clot. Thrombin is inactivated by circulating plasma heparin cofactor II (HCII),1 a member of the superfamily of serine * Corresponding author. Fax: +1 615 343 7023. Ingrid.Verhamme@vanderbilt.edu (I.M. Verhamme). 1 Abbreviations used: serpin, serine proteinase inhibitor; HCII, heparin cofactor II; AT, antithrombin; RCL, reactive center loop; SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; T, human α-thrombin; GAG, glycosaminoglycan; IPTG, isopropyl-1-thio-β-D-galactopyranoside; TNS, 2-(p-toluidinyl)naphthalene-6-sulfonic acid; DS, dermatan sulfate; CBS31.39, CH 3 SO 2 -D-LeuGly-Arg-p-nitroanilide; Chromozym TH, Tos-Gly-Pro-Arg-p-nitroanilide; pNA, p-nitroaniline; Hepes, N-(2-hydroxyethyl)-N′-2-piperazine-ethanesulfonic acid; PEG, polyethylene glycol 8000. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript proteinase inhibitors (serpins) [1][2][3]. Serpins possess a reactive center loop (RCL) that is cleaved by their target proteases, with formation of a stabilized, covalent serpin-enzyme complex in which the protease active site is distorted. The thrombin-HCII reaction is accelerated by binding of the sulfated glycosaminoglycans (GAGs), heparin and dermatan sulfate (DS) [4][5][6], and various other polyanions [7][8][9][10] in a ternary complex, and plays an important role in prevention of arterial thrombosis [11]. The thrombin-HCII mechanism utilizes an allosteric interaction of a unique acidic region in the HCII N-terminus with thrombin exosite I [1,...

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“…mg-' . cm-' at 280 nm) were taken as 65600 and 0.593 for HCII, 56600 and 0.624 for AT111 and 36600 and 1.75 for thrombin [5].…”

Section: Methodsmentioning

confidence: 99%

“…HCII (and ATIII) activity was determined by measuring the rate of thrombin inhibition in the absence and presence of either heparin or the phosphate-containing polyanions in 50 mM triethanolamine-acetate, 100 mM NaCl, 0.1% poly(ethyleneglycol) buffer at pH 8.0 and 25°C with at least a lo-fold molar excess of proteinase inhibitor to thrombin as described previously [5,14,15]. Inhibition rate constants were calculated as detailed [16].…”

Section: Assaysmentioning

confidence: 99%

Antithrombin action of phosvitin and other phosphate‐containing polyanions is mediated by heparin cofactor II

et al. 1988

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We have examined the antithrombin effects of various phosphate-containing polyanions (including linear polyphosphates, polynucleotides and the phosphoserine glycoprotein, phosvitin) on the glycosaminoglycan-binding plasma proteinase inhibitors, antithrombin III (ATIII) and heparin cofactor II (HCII). These phosphate-containing polyanions accelerate the HCII-thrombin reaction, as much as 1600-fold in the case of phosvitin. The HCII-thrombin reaction with both phosvitin and polynucleotides appears to follow the ternary complex mechanism. The HCII-thrombin complex is rapidly formed in the presence of these phosphate polyanions (each at 10 fig/ml) when 'ZSI-labeled thrombin is incubated with human plasma (ex vivo). None of these phosphate polyanions accelerate the ATIII-thrombin reaction. Our results suggest that the antithrombotic effect of these phosphate-containing polyanions is mediated by HCII activation and not by ATIII.

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Structure-function relationships in heparin cofactor II: Spectral analysis of aromatic residues and absence of a role for sulfhydryl groups in thrombin inhibition

Cited by 24 publications

References 37 publications

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli

Antithrombin action of phosvitin and other phosphate‐containing polyanions is mediated by heparin cofactor II

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