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

Abstract

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 ...

The Function and Metabolism of Vitamin K

“…This indicates that the extra "(-carboxyl groups of normal prothrombin are not available externally and are probably salt-linked to basic groups in the interior of the molecule. Munns et al (174) observed that confluent monolayers of H-35 hepatoma cells respond to vitamin K addition by the synthesis and secretion of mature prothrombin. As demonstrated by radioimmunassay, selective barium salt adsorption, and two coagulation assays that discriminate between precursor and mature prothrombin, these cells retained their ability to synthesize precur sor prothrombins in the absence of exogenous phylloquinone.…”

“…The 10 Gla residues in fragment I are at positions 7,8, 15,17,20,21,26,27,30, and 33 . "precursor forms". In fact, multiple intermediate forms of immature prothrom bin have been found in the endoplasmic reticulum of vitamin K-deficient rats (59,94,174).…”

The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

“…Previously published reports of in vitro studies have demon strated that vitamin K x treatment affects aspects of prothrombin synthesis in addition to γ-carboxylation. Vitamin K t treatment of rat hepatoma (H-35) cultures for 6 or 24 h resulted in a 2-or 5fold increase, respectively, in secreted prothrombin levels over control levels as measured by radioimmunoassay (13). Vit amin Kt treatment of human hepatoblastoma (HepG2) cultures for 24 h resulted in a 2-fold increase in secreted prothrombin over control levels as measured by radioimmunoassay (14).…”

The Prothrombin Gene

“…The amino-terminal region of the precursor sequence was highly hydrophobic and probably corresponds to the signal sequence found in other secreted Little is known about the regulation of the prothrombin gene. Munns et al [31] de scribed a rat hepatoma cell line that, in the presence of vitamin K, synthesized and se creted functionally active prothrombin. Graves et al [32,33] [34] have studied the hepatic microsomal forms of rat prothrombin.…”