Characterization of human, bovine, and horse antithrombin III

Kurachi, Kotoku; Schmer, G; Hermodson, Mark A.; Teller, David C.; Davie, Earl W.

doi:10.1021/bi00647a020

Cited by 168 publications

(73 citation statements)

References 37 publications

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“…The concentrations of purified proteins were determined using the extinction coefficients and molecular weights previously reported (6,10,11,(17)(18)(19)(20)(21)(22)(23).…”

Section: Methodsmentioning

confidence: 99%

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

Fuchs¹,

Trapp²,

Griffith³

et al. 1984

J. Clin. Invest.

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Abstract. The regulation of human Factor IX.was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, -60% of the '251-Factor IX8 was bound to antithrombin III (ATIII) by 2 h, with no binding to a2-macroglobulin or aI-proteinase inhibitor, as assessed by gel electrophoresis and IgG-antiproteinase inhibitor-Sepharose beads. In the presence ofheparin, virtually 100% ofthe 125I-Factor IXa was bound to ATIII by 1 min.

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“…The concentrations of purified proteins were determined using the extinction coefficients and molecular weights previously reported (6,10,11,(17)(18)(19)(20)(21)(22)(23).…”

Section: Methodsmentioning

confidence: 99%

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

Fuchs¹,

Trapp²,

Griffith³

et al. 1984

J. Clin. Invest.

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“…For some experiments, thrombin was inactivated at pH 7.5 with 1 mM DFP. An extinction coefficient of 5.7 (E2m) was used for antithrombin III (4).…”

Section: Methodsmentioning

confidence: 99%

“…F = (Lo -2KD, -Ro,) + \/(LO -2KDI -R0, )2 + 8LOKD, 4 Dividing by Lo, gives the fraction of the injected dose that is f'ree: F…”

Section: Appendixmentioning

confidence: 99%

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium

Lollar¹,

Owen²

1980

J. Clin. Invest.

197

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A B S T R A C T The clearance of 125I-thrombin and diisopropylphosphoryl-'251-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was -90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-'251-thrombin resulted in the rapid reappearance ofthe DIP-'25I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-1251-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to highaffinity binding sites, since dissociation constants (KD) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively.In contrast to DIP-thrombin, -75% of the radioacDr. Lollar is the recipient of National Institutes of Health grant P-32-HL-07344-2. Dr. Owen is the recipient of Career Development Award K04 HL 00348-03 from the National Heart, Lung, and Blood Institute.Received for publication 15 August 1979 and in revised form 11 August 1980. 1222 tivity associated with active thrombin remained in the circulation at 1 min. By 10 min 55% of 125I-thrombin had been removed from the circulation, and essentially all ofthe radioactivity can be accounted for in the liver. Sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of plasma samples taken after injection of '25I-thrombin demonstrated that all ofthe active thrombin was converted to covalent thrombin-antithrombin III complex by the time of initial sampling (30 s). The in vitro conversion of 125I-thrombin to thrombin-antithrombin III complex was considerably slower (50+5% conversion at 30 s). The simultaneous injection of excess unlabeled DIP-thrombin inhibited the rate of formation of '25I-thrombin-antithrombin III complex formation in vivo (but not in vitro), which suggests that the binding of active thrombin to the high affinity binding sites is required for the rapid inactivation ofthrombin in vivo.We propose that (a) thrombin in the circulation binds to active site-independent high-affinity binding sites on the endothelial cell surface; (b) the inactivation of thrombin by antithrombin III is faster in vivo than in vitro because the high-affin...

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“…Protein concentrations were determined using the following values for molecular weight and ⑀ 1 cm 1% at 280 nm, respectively: protein C, 62,000 and 14.5 (24); protein S, 75,000 and 9.5 (28); thrombin, 36,000 and 17.3 (29); and antithrombin III, 56,000 and 6.0 (30).…”

Section: Reagents-succinimidylmentioning

confidence: 99%

Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

Yegneswaran¹,

Smirnov²,

Safa³

et al. 1999

Journal of Biological Chemistry

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The effect of replacing the ␥-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming 2 ‫؍‬ 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 Å, compared with 94 Å for wildtype APC. The ␥-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 Å observed for the APC⅐protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg 306 , but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC⅐protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg 306 . Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.

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Characterization of human, bovine, and horse antithrombin III

Cited by 168 publications

References 37 publications

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium

Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

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