The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

Summary

Whether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VII a and assessed its effect on factor VII: tissue factor interactions. FVII-GP inhibited the activation of factor X by factor Vila in the presence of either soluble or cell surface tissue factor halfmaximally at 0.5 μM and 2.7 μM, respectively. However, FVII-GP failed to inhibit the specific binding of factor Vila to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor Vila. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor Vila to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VII a was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206. The antibody reacted with Gla-domainless factor Vila, but only 37% as compared to intact factor Vila. In addition, PW4 as well as its Fab’ fragment, inhibited factor Vila binding to cell-surface tissue factor. These studies indicate that the factor VII Gla-domain does not provide structural elements that contribute to the formation of a stable factor VII/VII a-tissue factor binary complex. The factor VII Gla-domain appears to be necessary, however, in binding calcium ions and inducing a calcium-dependent conformational change in factor VII/VII a that expresses one or more neoepitopes that participates in the interaction of factor VII/VII a with the extracellular domain of tissue factor apoprotein.

• References

• 1 Hagen FS, Gray CL, O’Hara P, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley M, Kisiel W, Kurachi K, Davie EW. Caracterization of a cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986; 83: 2412-2416
• 2 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71: 1-7
• 3 Bach R, Rifkin DB. Expression of tissue factor procoagulant activity: Regulation by cytosolic calcium. Proc Natl Acad Sci USA 1990; 87: 6995-6999
• 4 Sakai T, Lund-Hansen T, Thim L, Kisiel W. The ³-carboxyglutamic acid domain of factor VII a is essential for its interaction with cell surface tissue factor. J Biol Chem 1990; 276: 1890-1894
• 5 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976; 72: 248-254
• 6 Laemmli UK. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 7 Wright SF, Bourne CD, Hoke RA, Koehler KA, Hiskey RG. A method for specifi modification of ³-carboxyglutamic acid residues in proteins. Anal Biochem 1984; 139: 82-90
• 8 Thim L, Hansen MT, Sdrensen AR. Secretion of human insulin by a transformed yeast cell. FEBS Lett 1987; 212: 307-312
• 9 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide, l,3,4,6-tetrachloro-3a, 6a-diphenylgly-couril. Biochem Biophys Res Commun 1978; 80: 849-857
• 10 Thim L, Bjoern S, Christensen M, Nicolaisen EM, Lund-Hansen T, Pedersen AH, Hedner U. Amino acid sequence and posttranslational modifications of human factor VII a from plasma and transfected baby hamster kidney cells. Biochemistry 1988; 27: 7785-7793
• 11 Kondo S, Kisiel W. Evidence that plasma lipoproteins inhibit the factor VII a-tissue factor complex by a different mechanisms than extrinsic pathway inhibitor. Blood 1987; 70: 1947-1954
• 12 Stem DM, Nawroth PP, Kisiel W, Handley D, Drillings M, Bantos J. A coagulation pathway on bovine aortic segments leading to generation of factor Xa and thrombin. J Clin Invest 1984; 74: 1910-1921
• 13 Wildgoose P, Kisiel W. Activation of human factor VII by factors IX a and Xa on human bladder carcinoma cells. Blood 1989; 73: 1888-1895
• 14 Pedersen AH, Nordfang O, Norris F, Wiberg FC, Christensen PM, Moeller KB, Meidal-Pedersen J, Beck TC, Norris K, Hedner U, Kisiel W. Recombinant human extrinsic pathway inhibitor. J Biol Chem 1990; 265: 16786-16793
• 15 Wildgoose P, Berkner KL, Kisiel W. Synthesis, purification, and characterization of an Argl52 Glu site directed mutant of recombinant human blood clotting factor VII. Biochemistry 1990; 29: 3413-3420
• 16 Komiyama Y, Pedersen AH, Kisiel W. Proteolytic activation of human factors IX and X by recombinant human factor Vila. Effects of calcium, phospholipids, and tissue factor. Biochemistry 1990; 29: 9148-9125
• 17 Bach R, Gentry R, Nemerson Y. Factor VII binding to tissue factor in reconstituted phospholipid vesicles: Induction of cooperativity by phosphatidylserine. Biochemistry 1986; 25: 4007-4020
• 18 Wildgoose P, Kazim AL, Kisiel W. The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor. Proc Natl Acad Sci USA 1990; 87: 7290-7294
• 19 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predifined specificity. Nature 1975; 256: 495-497
• 20 Kazen A, Glick M. Oxytocin, Methods of Hormone Radioimmunoassay. Jaffe and Behrmann; New York: 1979. p 328
• 21 Akiyama H, Sinha D, Seaman FS, Kirby EP, Walsh PN. Mechanism of activation of coagulation factor XI by factor XII a studied with monoclonal antibodies. J Clin Invest 1986; 78: 1631-1636
• 22 Ruf W, Rehemtulla A, Edgington TS. Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem 1991; 266: 2158-2166
• 23 Nemerson Y. The phospholipid requirement of tissue factor in blood coagulation. J Clin Invest 1968; 47: 72-80
• 24 Gerads I, Govers-Riemslag JWP, Tans G, Zwaal RFA, Rosing J. Prothrombin activation on membranes with anionic lipids containing phosphate, sulphate, and/or carboxyl groups. Biochemistry 1990; 29: 7967-7974
• 25 Wildgoose P, Kisiel W. Inhibition of prothrombin activation by factor X and factor IX Gla-peptides. Biochem Biophys Res Comm 1988; 152: 1207-1212
• 26 Nawroth PP, Kisiel W, Stem DM. Anticoagulant and antithrombotic properties of a ³-carboxyglutamic acid-rich peptide derived from the light chain of blood coagulation factor X. Thromb Res 1986; 44: 625-637
• 27 Rao LVM, Rapaport SI, Tait JF. Role of negatively charged phospholipids in Vila- catalyzed activation of FX on the cell surface. Circulation 1990; 82: III-132 (abstract)