Differential Effects of Warfarin on the Intracellular Processing of Vitamin K-dependent Proteins

Summary

The vitamin K-dependent carboxylation of specific glutamyl residues to γ-carboxyglutamyl residues occurs during the endoplasmic reticulum processing of a limited number of proteins. The fate of the under-γ-carboxylated proteins during protein processing was studied. When human hepatoma (HepG2) cells were grown in the presence of warfarin, under-γ-carboxylated prothrombin was secreted into the medium. In contrast, prothrombin secretion from a rat hepatoma (H-35) cell line was blocked by warfarin, and intracellular forms which were retained were degraded. When rat prothrombin (rFII) was stably transfected into warfarin treated HepG2 cells, endogenous human prothrombin (hFII) was secreted in an under-γ-carboxylated form, while rFII accumulated intracellularly. These data indicate that retention and degradation of under-γ-carboxylated prothrombin by human hepato-cytes is related to a structural difference in rFII and hFII. When rFII and hFII were transfected into a warfarin treated transformed human embryonic kidney cell line (293), both proteins were secreted in an under-γ-carboxylated form and intracellular retention was not observed. However, the secretion of rFII was greatly diminished. Cellular retention of under-γ-carboxylated forms is therefore tissue specific, but degradation is not.

• References

• 1 Suttie JW. Synthesis of vitamin K-dependent proteins. FASEB J 1993; 7: 445-452
• 2 Furie B, Furie BC. Molecular and cellular biology of blood coagulation. N Engl J Med 1992; 326: 800-806
• 3 Suttie JW. Vitamin K Antagonists. In: Hemostasis and Thrombosis. Basic Principles and Clinical Practice Colman RW, Hirsh J, Marder VJ, Salzman EW. eds J. B. Lippincott Company; Philadelphia, PA: 1994: 1562-1566
• 4 Stenfio J. Dicumarol-induced prothrombin in bovine plasma. Acta Chem Scand 1970; 24: 3762-3763
• 5 Ganrot PO, Nilehn JE. Plasma prothrombin during treatment with Dicuma-rol. II. Demonstration of an abnormal prothrombin fraction. Scand J Clin Lab Invest 1968; 22: 23-28
• 6 Carlisle TL, Shah DV, Schlegel R, Suttie JW. Plasma abnormal prothrombin and microsomal prothrombin precursor in various species. Proc Soc Exp Biol Med 1975; 148: 140-144
• 7 Shah DV, Swanson JC, Suttie JW. Abnormal prothrombin in the vitamin K-deficient rat. Thrombosis Res 1984; 35: 451-458
• 8 Yamanaka Y, Yamano M, Yasunaga K, Shike T, Uchida K. Effect of warfarin on plasma and liver vitamin K levels and vitamin K epoxide reductase activity in relation to plasma clotting factor levels in rats. Thrombosis Res 1990; 47: 205-214
• 9 Shah DV, Suttie JW. Mechanism of action of vitamin K: Evidence for the conversion of a precursor protein to prothrombin in the rat. Proc Natl Acad Sci USA 1971; 68: 1653-1657
• 10 Zhang P, Suttie JW. Prothrombin synthesis and degradation in rat hepatoma (H-35) cells: Effects of warfarin. Blood 1994; 84: 169-175
• 11 Bolger R. Processing of the Vitamin K-dependent Proteins. Ph.D. Thesis, Univ Wisconsin-Madison 1993: 197
• 12 Wallin R. The effects of warfarin on HepG2 cells suggest that prothrombin and factor X interact differently with the vitamin K-dependent carboxylase in the secretory pathway. Thrombosis Res 1991; 62: 235-240
• 13 Wallin R, Martin LF. Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase. J Biol Chem 1988; 263: 9994-10001
• 14 Fair DS, Bahnak BR. Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III. Blood 1984; 64: 194-204
• 15 Fair DS, Marlar RA. Biosynthesis and secretion of factor VII, protein C, protein S, and the protein C inhibitor from a human hepatoma cell line. Blood 1986; 67: 64-70
• 16 McClure DB, Walls JD, Grinnell BW. Post-translational processing events in the secretion pathway of human protein C, a complex vitamin K-depen-dent antithrombotic factor. J Biol Chem 1992; 267: 19710-19717
• 17 Miletich JP, Broze Jr GJ, Majems PW. The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II, IX, and LX. Analyt Biochem 1980; 105: 304-310
• 18 Swanson JC, Suttie JW. Prothrombin biosynthesis: Characterization of processing events in rat live microsomes. Biochemistry 1985; 24: 3890-3897
• 19 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt Biochem 1987; 162: 156-159
• 20 Dihanich M, Monard D. cDNA sequence of rat prothrombin. Nucleic Acids Res 1990; 18: 4251
• 21 Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973; 52: 456-461
• 22 Schaffer JE, Lodish HF. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein. Cell 1994; 79: 427-436
• 23 Berkner KL, Sharp PA. Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant. Nucleic Acids Res 1985; 13: 841-857
• 24 D’Angelo A, Vigano-D’Angelo S, Esmon CT, Comp PC. Acquired deficiencies of protein S, protein S activity during oral anticoagulation, in liver disease, and in disseminated intramuscular coagulation. J Clin Invest 1988; 81: 1445-1454
• 25 Bertina RM, Wijngaarden Av, Reinalda-Poot J, Poort SR, Bom VJJ. Determination of plasma protein S - the protein cofactor of activated protein C. Thromb Haemost 1985; 53: 268-272
• 26 Epstein DJ, Bergum PW, Bajaj SP, Rapaport SI. Radioimmunoassays for protein C and factor X. Plasma antigen levels in abnormal hemostatic states. Am J Clin Pathol 1984; 82: 573-581
• 27 Bonifacino JS, Lippincott-Schwarts J. Degradation of proteins within the endoplasmic reticulum. Curr Opin Cell Biol 1991; 3: 592-600
• 28 Rajagopalan S, Xu Y, Brenner MB. Retention of unassembled components of integral membrane proteins by calnexin. Science 1994; 263: 387-390
• 29 David V, Hochstenbach F, Rajagopalan S, Brenner MB. Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). J Biol Chem 1993; 268: 9585-9592