Large-Scale Preparation of Human Thrombin

Kaetsu, Hiroshi; Mizuguchi, Jin; Hamamoto, Takayoshi; Kamimura, Koichiro; Yoshida, Yasuko; Nakagaki, Tomohiro; Ogata, Youichi; Miyamoto, Seiji; Funatsu, Asami

doi:10.1016/s0049-3848(98)00026-7

Cited by 5 publications

(1 citation statement)

References 17 publications

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“…Recombinant human soluble TF (sTF; residues 1-218) was a gift from Dr. Toshiyuki Miyata (National Cardiovascular Center, Suita, Japan), and it was prepared as described (25). Human plasma-derived thrombin, activated protein C, and FIXa were gifts from Hiroshi Kaetsu, Shinji Nakahira, and Takayoshi Hamamoto (KAKETSUKEN), respectively, and prepared as described (23,26,27). Three cardiotoxins (CM-14, CM-17, and CM-18 from Naja naja atra) were obtained from Dr. Mitsuhiro Ohta (Kobe Pharmaceutical University).…”

Section: Proteinsmentioning

confidence: 99%

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Banerjee

Mizuguchi

Iwanaga

et al. 2005

Journal of Biological Chemistry

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During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF⅐FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF⅐FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF⅐FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF⅐FVIIa amidolytic activity, with a K i of 50 nM. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 ؋ 10 5 M ؊1 . The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF⅐FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

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Section: Proteinsmentioning

confidence: 99%

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Banerjee

Mizuguchi

Iwanaga

et al. 2005

Journal of Biological Chemistry

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Large-scale preparation of thrombin from human plasma

Aizawa¹,

Winge²,

Karlsson³

2008

Thrombosis Research

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Domain structure of bi-functional selenoprotein P

Saito

Sato

Hirashima

et al. 2004

Biochemical Journal

113

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Human selenoprotein P (SeP), a selenium-rich plasma glycoprotein, is presumed to contain ten selenocysteine residues; one of which is located at the 40th residue in the N-terminal region and the remaining nine localized in the C-terminal third part. We have shown that SeP not only catalyses the reduction of phosphatidylcholine hydroperoxide by glutathione [Saito, Hayashi, Tanaka, Watanabe, Suzuki, Saito and Takahashi (1999) J. Biol. Chem. 274, 2866-2871], but also supplies its selenium to proliferating cells [Saito and Takahashi (2002) Eur. J. Biochem. 269, 5746-5751]. Treatment of SeP with plasma kallikrein resulted in a sequential limited proteolysis (Arg-235-Gln-236 and Arg-242-Asp-243). The N-terminal (residues 1-235) and C-terminal (residues 243-361) fragments exhibited enzyme activity and selenium-supply activity respectively. These results confirm that SeP is a bi-functional protein and suggest that the first selenocysteine residue is the active site of the enzyme and the remaining nine residues function as a selenium supplier.

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Large-Scale Preparation of Human Thrombin

Cited by 5 publications

References 17 publications

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Large-scale preparation of thrombin from human plasma

Domain structure of bi-functional selenoprotein P

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