The preparation of activated Factor X and its action on prothrombin

Jesty, Jolyon; Esnouf, M. P.

doi:10.1042/bj1310791

Cited by 86 publications

(37 citation statements)

References 22 publications

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“…Human FXa, human FX, and human FVIIa were purchased from Innovative Research (Novi, MI) and bovine FXa was purified from bovine plasma (27). Protein S was from Hematologic Technologies (Essex Junction, VT).…”

Section: Methodsmentioning

confidence: 99%

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

Dockal

Hartmann

Fries

et al. 2014

Journal of Biological Chemistry

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Background: Tissue factor pathway inhibitor (TFPI) inhibits coagulation factors Xa and VIIa. Results: A de novo synthesized 20-mer peptide that binds to TFPI was structurally and functionally characterized. Conclusion:The peptide binds to the Kunitz domain 1 of TFPI and blocks inhibition of factor Xa and factor VIIa by TFPI. Significance: The peptide can potentially prevent bleeding in hemophilia patients.

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Section: Methodsmentioning

confidence: 99%

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

Dockal

Hartmann

Fries

et al. 2014

Journal of Biological Chemistry

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“…E. coli OM proteins OmpA, FepA, and FepA⌬13-17 were purified by differential extraction of cell envelopes with Triton X-100 (77,91). Colicin B (ColB) was derived from supernatants of E. coli DM1187/pCol1, and factor X was from bovine blood; we purified both the toxin (77) and the protease (105) by conventional chromatographic methods and activated factor X to factor Xa with snake venom (45). E. coli MalE was purified from E. coli ER2507/pMalp2 after IPTG induction by French pressure cell lysis and subsequent amylose chromatography (32).…”

Section: Methodsmentioning

confidence: 99%

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

et al. 2008

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We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB ؉ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepA⌬3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.Iron is one target of gram-negative bacterial cell envelope transport systems, and microbes elaborate high-affinity siderophores that complex extracellular iron (70). However, ferric siderophores, like ferric enterobactin (FeEnt), are too large (716 Da) to pass through general porins in the outer membrane (OM), necessitating a different type of transporter to acquire them. On the basis of their 22-stranded transmembrane ␤-barrels, OM metal transporters like FepA belong to the porin superfamily (89). Ligand binding to such receptors initiates the transport reaction through their transmembrane channels, which led to their designation as ligand-gated porins (LGP) (88), by analogy to the family of eukaryotic ligand-gated ion channels. It is noteworthy that LGP are mechanistically distinct from general, diffusive porins because they bind metal complexes with high affinity and actively transport them against a concentration gradient into the cell. Once in the periplasm, binding proteins adsorb ferric siderophores and del...

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“…To this, Russell's viper venom was added at a concentration of 1:200 (venom: protein) and activation was performed by incubating the sample stirring at 37°C for 3 hours. [4,[7][8][9].…”

Section: Isolation Of Prothrombin Rich Fraction and Activationmentioning

confidence: 99%

“…Conventionally thrombin has been prepared from bovine prothrombin derived from bovine plasma [4,5]. The prothrombin is converted to thrombin using thromboplastin in presence of calcium chloride ions or by snake venom enzymes [6].…”

Section: Introductionmentioning

confidence: 99%

Rapid purification of high purity thrombin and preparation of a novel hemostat for clinical purposes

Turaga

Rao

Sripad

2008

Indian J Hematol Blood Transfus

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Thrombin was prepared from crude prothrombin enriched plasma by activation using Russell's viper venom. Prothrombin was prepared by barium sulphate adsorption and elution of prothrombin enriched fraction using high concentrations of sodium citrate. This fraction was directly activated with venom and thrombin was purifi ed by SP Sepharose ion-exchange chromatography and subsequently over Phenyl-sepharose column. This product exhibits a purity of >98% with an activity of at least 6000U/ mg or higher. The Thrombin was further used in the preparation of a novel bio-absorbable hemostat using Calcium Alginate fi ber sheet which acts as an absorbable hemostat. The present hemostat is highly porous, easy to use and has faster clotting time due to higher solubility of calcium fi bers and thereby releasing thrombin.

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The preparation of activated Factor X and its action on prothrombin

Cited by 86 publications

References 22 publications

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

Rapid purification of high purity thrombin and preparation of a novel hemostat for clinical purposes

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