Mechanism of Action of Vitamin K: Evidence for the Conversion of a Precursor Protein to Prothrombin in the Rat

Shah, D.V.; Suttie, John W.

doi:10.1073/pnas.68.7.1653

Cited by 68 publications

(23 citation statements)

References 27 publications

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“…These hypotheses include regulation at the levels of transcription (3), translation (4)(5)(6), and, more recently, at the post-translational level (7)(8)(9)(10). During the past several years, much attention has been focused on the possibility that the inactive "abnormal" prothrombins (11)(12)(13) present in animals deficient in vitamin K or receiving coumarin anticoagulants may represent an unmodified species of mature prothrombin, i.e., precursor prothrombin.…”

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confidence: 99%

“…Because preliminary studies with Nonidet P-40 indicated that this detergent inhibited the clotting activity associated with the assay of Shapiro and Waugh (24) (but not that of the Echis carinatus assay), the prothrombin contained in 105,000 X g supernatant fractions was initially adsorbed to barium sulfate (7,25) prior to assessing clotting activity. Adsorption was conducted as previously described (7,25) barium treatment (see Tables 1 and 2). Thus, the ability to discriminate between preprothrombin and prothrombin in various cell and medium fractions was based not only on two distinct clotting assays, but also on the well-established ability of prothrombin to be selectively adsorbed to insoluble barium salts.…”

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“…Two coagulation assays were used to discriminate between prothrombin and preprothrombin. These included the Echis carinatus assay (7,13), and the physiological two-stage assay method of Shapiro and Waugh (24) employing thromboplastin. Since the Echis carinatus assay was capable of generating clotting activity from both preprothrombin and prothrombin preparations while the two-stage assay generated a clotting activity only from prothrombin (13,25), employment of both assays provided a means of assessing the preprothrombin and prothrombin content of various cell and media fractions.…”

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Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells.

Munns¹,

Johnston²,

Liszewski³

et al. 1976

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

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The ability of confluent monolayers of H-35 cells, originally obtained from a rat hepatoma, to synthesize prothrombin in response to vitamin K1 (phylloquinone) was studied. As demonstrated by radioimmunoassay, selective barium salt adsorption, and two coagulation assays which discriminate between precursor-and mature-prothrombin, these cells retained their ability to synthesize precursor prothrombin (preprothrombin) in the absence of exogenous phylloquinone (vitamin K). When phylloquinone was added to the medium (100 ng/ml), the existing intracellular concentration of preprothrombin was reduced to 50% within 1 hr after exposure to the vitamin and slowly declined thereafter to approximately 30% of control levels by 36 hr. Concomitant with the rapid loss of intracellular pre rothrombin was the appearance of mature prothrombin in the medium. The appearance of prothrombin was biphasic: occurring during the initial 0-6 hr interval, and again at an increased rate during the next 18-24 hr interval. The amount of prothrombin appearing in the medium exceeded by severalfold the amount of precursor mobilized. These data demonstrate that monolayer cultures of H-35 hepatoma cells retain their ability to synthesize preprothrombin and other enzymes, responsible for post-translational modification of prothrombin and its subsequent secretion, under the influence of vitamin K.Numerous hypotheses have been advanced regarding the mechanism by which vitamin K (phylloquinone) catalyzes prothrombin biosynthesis (1, 2). These hypotheses include regulation at the levels of transcription (3), translation (4-6), and, more recently, at the post-translational level (7-10). During the past several years, much attention has been focused on the possibility that the inactive "abnormal" prothrombins (11-13) present in animals deficient in vitamin K or receiving coumarin anticoagulants may represent an unmodified species of mature prothrombin, i.e., precursor prothrombin. Evidence to support this view includes the presence and absence of y-carboxyglutamate residues in prothrombin and preprothrombin, respectively (14-16), the ability of inactive prothrombin to be converted to thrombin by Echis carinatus venom (8, 17), the in vitro conversion of preprothrombin to prothrombin by rat liver microsomes (18), and the vitamin K-dependent incorporation of 14CO2 into prothrombin from liver microsomes of vitamin K-deficient rats (10).In the present communication, we describe a hepatoma cell culture system which is suitable to investigate the vitamin Kdependent synthesis and/or modification of prothrombin. We present data which support the hypothesis that the conversion of preprothrombin to prothrombin in H-35 hepatoma cells is vitamin K-dependent. Further, these data indicate that after this conversion an acceleration in the overall rate of synthesis of prothrombin occurs which suggests regulatory controls for both translation and transcription of preprothrombin Radioimmunoassay of Prothrombin. The radioimmunoassay for prothrombin was performed as ...

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Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells.

Munns¹,

Johnston²,

Liszewski³

et al. 1976

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

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“…In humans, VKD-␥-carboxylases change glutamic acid residues in blood clotting factors into ␥-carboxylated glutamic acid (35). VKD-␥-carboxylase requires epoxidation of vitamin K for this modification (36). The SdpB homology to VKD-␥-carboxylases is restricted mostly to the N-terminal portion of VKD-␥-carboxylases specifically from amino acids 13 to 283 (34).…”

Section: Discussionmentioning

confidence: 99%

Production of the Cannibalism Toxin SDP Is a Multistep Process That Requires SdpA and SdpB

Morales

Liu

et al. 2013

Journal of Bacteriology

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bDuring the early stages of sporulation, a subpopulation of Bacillus subtilis cells secrete toxins that kill their genetically identical siblings in a process termed cannibalism. One of these toxins is encoded by the sdpC gene of the sdpABC operon. The active form of the SDP toxin is a 42-amino-acid peptide with a disulfide bond which is processed from an internal fragment of pro-SdpC. The factors required for the processing of pro-SdpC into mature SDP are not known. We provide evidence that pro-SdpC is secreted via the general secretory pathway and that signal peptide cleavage is a required step in the production of SDP. We also demonstrate that SdpAB are essential to produce mature SDP, which has toxin activity. Our data indicate that SdpAB are not required for secretion, translation, or stability of SdpC. Thus, SdpAB may participate in a posttranslation step in the production of SDP. The mature form of the SDP toxin contains a disulfide bond. Our data indicate that while the disulfide bond does increase activity of SDP, it is not essential for SDP activity. We demonstrate that the disulfide bond is formed independently of SdpAB. Taken together, our data suggest that SDP production is a multistep process and that SdpAB are required for SDP production likely by controlling, directly or indirectly, cleavage of SDP from the pro-SdpC precursor.

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“…Moreover, the protein was electrophoretically distinguishable from normal prothrombin [11]. Very soon thereafter, in another insightful experiment, Shah and Suttie [13] injected vitamin K-deficient rats with radioactive amino acids and showed that the action of vitamin K was to convert an inactive precursor version of prothrombin to an active one. …”

Section: The Vitamin K Storymentioning

confidence: 99%

Some Important Milestones in the Field of Blood Clotting

Doolittle¹

2015

J Innate Immun

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Several different kinds of ‘milestone' in the field of blood coagulation are described from the middle decades of the 20th century. Although viewed from the standpoint of clotting per se, attention is also given to implications for innate immunity. The first milestone considered is the protracted saga of clotting dependence on vitamin K, an adventure that spanned more than five decades beginning in the 1920s. The second has to do with the discovery of a half-dozen ‘new' clotting factors during the period immediately following World War II. A third pursues a narrower focus and examines the once mysterious transformation of fibrinogen into fibrin. Finally, the clinical treatment of classical hemophilia had a remarkable turning point in the 1960s as the result of simple but sensible measures.

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Mechanism of Action of Vitamin K: Evidence for the Conversion of a Precursor Protein to Prothrombin in the Rat

Cited by 68 publications

References 27 publications

Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells.

Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells.

Production of the Cannibalism Toxin SDP Is a Multistep Process That Requires SdpA and SdpB

Some Important Milestones in the Field of Blood Clotting

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