Abstract
Factor X in plasma is a gamma-carboxylated two-chain glycoprotein which, in activated form, plays a pivotal role in blood coagulation. We have utilized purified rat Factor X antibody, coupled to Sepharose, to isolate and characterize Factor X in rat liver, plasma, and hepatoma cells. Rat factor X is synthesized as a single chain precursor (Mr = 63,000). It is this form which undergoes vitamin K-dependent carboxylation in rat liver microsomes. Only after secretion is Factor X converted into its two-chain mature form. Single chain X synthesis and secretion in hepatoma cells is enhanced by vitamin K. The amount of single chain X secreted by these cells is one-half that of prothrombin. The NH2-terminal gamma-carboxylated fragments of prothrombin which induce prothrombin synthesis (Graves, C. B., Munns, T. W., Carlisle, T. L., Grant, G. A., and Strauss, A. W. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4772-4776) also induce single chain X synthesis by hepatoma cells. We propose that synthesis of all vitamin K-dependent proteins may be regulated by this common control mechanism.
Highlights
Glycoprotein which, in activated form, plays a pivotal Factor Xa is the active proteolytic enzymeformed from role in blood coagulationW. e have utilized purified rat Factor X which, in the presence of Factor Va, calcium, and Factor X antibody, coupledto Sepharose,to isolate and phospholipid, converts prothrombin to thrombin
The results showed that NHz-terminal fragmentsof carboxylated fragments of prothrombin which induce prothrombincontainingpeptide-bound y-carboxyglutamyl prothrombin synthesis
Undertheseconditionsthe quantity of secreted Factor X was nearly identical with the values exhibited by vitamin-depleted-cells. These results indicate that the inhibition of proteolytic enzymes blocks vitamin K-dependent inductionof Factor X synthesis
Summary
The other vitamin K-dependent proteins (prothrombin, Factors VI1 and IX, and proteins C, S, and Z ). Cell Culture System-The cell line H4-ll-E-C3 (ATCC CRL1548, Reuber H-35 rat minimal deviation hepatoma) used in these studies of Factor X has significant homology to the catalytircegion of was grown and maintainedinmonolayer culture as previousl-.,v d-~e~-. These same vitamin K-dependent proteins antdo other serine scribid (11). ["HlLeucinelabeled Factor X was adsorbed to and eluted from Sepharose-conjugated antibodies to rat FactXoraccording to the procedures described for the isolation of"H-labeled rat prothrombin(11). K (0.5 pg/ml) was added for 1.5 h prior to refeeding cells with fresh medium containing vitamin K (0.1 pg/ml) and 0.13 p~ bovine prothrombin fragment 1. Assay for Factor X Activity-Rat plasma was assayed for Factor X activity using Factor VI1 and X-deficient bovine plasma according to the method describedby Bachman et al (21)
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