Isolation and purification of bovine and canine prothrombin

Moore, H.C.; Lux, Samuel E.; Malhotra, O.P.; Bakerman, Seymour; Carter, J.R.

doi:10.1016/0304-4165(65)90484-8

Cited by 43 publications

(8 citation statements)

References 11 publications

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“…Under the same conditions, diluted plasma proteins were found to adsorb to an extent of 20 %. This low adsorption of non-prothrombin proteins would probably be removed by washing the barium citrate during the normal isolation procedure for prothrombin (Moore et al, 1965;Nelsestuen and Suttie, 1972a).…”

Section: Resultsmentioning

confidence: 99%

Mode of action of vitamin K. Calcium binding properties of bovine prothrombin

Nelsestuen

Suttie²

1972

Biochemistry

138

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Section: Resultsmentioning

confidence: 99%

Mode of action of vitamin K. Calcium binding properties of bovine prothrombin

Nelsestuen

Suttie²

1972

Biochemistry

138

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“…Preparation of canine prothrombin complex. To prepare the canine prothrombin complex, pooled citrated canine plasma containing 2 mM benzamidine was used and the prothrombin complex was partially purified by barium citrate adsorption, sodium citrate wash, and EDTA elution (27). The partially purified complex was treated with diisopropylfluorophosphate (2 mM final concentration) and dialyzed extensively against 20 mM Tris buffer, pH 7.4, containing 150 mM NaCI.…”

Section: Methodsmentioning

confidence: 99%

Activation of Protein C In Vivo

1982

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“…Rat PT was purified as described (14). Bovine PT, fragment 1 (residues 1-156), fragment 2 (residues 157-274), and thrombin (residues 275-582) were prepared as reported (16) from a barium citrate precipitate (17) except that chromatography on heparin linked to agarose preceded gel filtration in the PT preparation. Residues 1-42 and 46-156 were prepared according to Morita and Jackson (18).…”

Section: Methodsmentioning

confidence: 99%

Induction of prothrombin synthesis by prothrombin fragments.

Graves¹,

Munns²,

Carlisle³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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The mechanisms by which blood levels of prothrombin (PT) are regulated in the vitamin K-sufficient state are unknown. We have studied PT synthesis by Reuber H-35 rat hepatoma cells exposed to vitamin K and [3H]leucine in serum-free cultures. Administration to the culture system ofexogenous bovine PT and rat PT was characterized by increases in endogenous PT synthesis and secretion of'2-. and 3-fold, respectively. This induction required endogenous proteolytic degradation of PT. Studies conducted with bovine PT fragment 1 (residues 1-156) demonstrated up to 5-fold increases in PT synthesis. This induction was dose dependent and saturable. Addition of bovine PT chymotryptic fragments to the cells indicated that the NH2-terminal peptide of prothrombin (residues 1-42) contained the requisite structural elements for the induction. Peptide-bound y-carboxyglutamate residues were required for the observed stimulation of'PT synthesis. These results suggest that PT synthesis might be regulated physiologically by the products formed during its normal turnover and consumption during blood coagulation. Normal coagulation requires that plasma concentrations of the clotting factors be maintained within relatively narrow ranges. An understanding of the mechanisms by which their synthesis and consumption are regulated is therefore essential. Extensive structural and enzymatic studies of several clotting factors (1, 2) have provided information concerning their activation and, thus, consumption. However,, only recently has the regulation of their biosynthesis been investigated (3-10). While the essential role of vitamin K in the 'y-carboxylation of glutamic acid residues in clotting factors II, VII, IX, and Xhas been elucidated (10), the mechanism by which the biosynthesis of these and other clotting factors are regulated when sufficient vitamin K is, present is poorly understood. Several investigators (3-5) have reported that a factor present in the plasma of hypoprothrombinemic animals will induce synthesis of the vitamin K-dependent coagulation factors in normal animals. Other workers have suggested that synthesis of fibrinogen (7, 8) or prothrombin (11) is regulated by polypeptides generated from these factors during normal coagulation. Kessler and coworkers (7,8) found that, after infusions of fibrin degradation products, fibrinogen synthesis was increased. Unfortunately, a comparable study (9) Because ofthe central role ofPT in coagulation, we have continued our studies on regulation of its biosynthesis (11). Prothrombin, the plasma zymogen of thrombin, is a single polypeptide chain with an apparent molecular weight of 68,000 to 74,000. The complete amino acid sequences of bovine and human PT and parts of the rat protein are known (12)(13)(14). During coagulation, thrombin is generated from PT by the action of Factor Xain the presence ofFactor Va, Ca2+, and phospholipids. While the NH2-terminal, y-carboxyglutamyl-containing portion (fragment 1.2) of PT is required for optimal physiological activation (1, 2), the f...

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Isolation and purification of bovine and canine prothrombin

Cited by 43 publications

References 11 publications

Mode of action of vitamin K. Calcium binding properties of bovine prothrombin

Mode of action of vitamin K. Calcium binding properties of bovine prothrombin

Activation of Protein C In Vivo

Induction of prothrombin synthesis by prothrombin fragments.

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