Calcium Ion-Dependent Monoclonal Antibody Against Human Fibrinogen: Preparation, Characterization, and Application to Fibrinogen Purification

Summary

We have produced a high-affinity monoclonal antibody classified as IgG₁ with K-type light chains that recognizes the calcium ion(Ca²⁺)- dependent conformation of the D-domain of human fibrinogen. Binding of fibrinogen in solution to the insolubilized antibody increased in the presence of increasing concentrations of up to 2 mM Ca²⁺, the half-maximal binding being reached at 130 μM Ca²⁺. The dissociation constant was estimated to be 1.6 × 10^-8 M at 2 mM Ca²⁺. The antibody was found also to be dependent on other divalent metal ions including Zn²⁺, Mn²⁺, Co²⁺ and Cu²⁺, but not Ba²⁺, Mg²⁺ or Sr²⁺. The synthetic Gly-Pro-Arg-Pro-amide peptide, which has recently been shown to bind to close proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this antibody. This antibody was found to be a suitable ligand for the immunoaffinity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or heparinized plasma by eliminating the precipitation procedure widely adopted in conventional techniques of fibrinogen purification. Indeed, fibrinogen Marburg I with the Aa chains depleted of the carboxy-terminal Aα(461-610) residue segment has been purified by this technique, although this dysfibrinogen was difficult to purify by conventional precipitation techniques.

• References

• 1 Blombäck B, Blombäck M. The molecular structure of fibrinogen. Ann NY Acad Sci 1972; 202: 77-79
• 2 Doolittle RF. Structural aspects of the fibrinogen-fibrin conversion. Advances in Protein Chem 1973; 27: 1-109
• 3 Doolittle RF. Fibrinogen and fibrin. Ann Rev Biochem 1984; 53: 195-229
• 4 Fowler WE, Erickson HP. Trinodular structure of fibrinogen. Confirmation by both shadowing and negative stain electron microscopy J Mol Biol 1979; 134: 241-249
• 5 Weisel JW, Stauffacher CV, Bullitt E, Cohen C. A model of fibrinogen: domains and sequence. Science 1985; 230: 1388-1391
• 6 Haverkate F, Timan G. Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin products D. Thromb Res 1977; 10: 803-812
• 7 Boyer MH, Shainoff JR, Ratnoff OD. Acceleration of fibrin polymerization by calcium ions. Blood 1972; 39: 382-387
• 8 Endres GF, Scheraga HA. Effects of calcium ions on the reversible polymerization of fibrin monomer. Arch Biochem Biophys 1972; 153: 266-278
• 9 Brass EP, Forman WB, Edwards RV, Lindan O. Fibrin formation: Effect of calcium ions. Blood 1978; 52: 654-658
• 10 Nieuwenhuizen W, van Ruijven-Vermeer IA M, Nooijen WJ, Vermond A, Haverkate F, Hermans J. Recalculation of calcium-binding properties of human and rat fibrin(ogen) and their degradation products. Thromb Res 1981; 22: 653-657
• 11 Marguerie G, Chagnel G, Suscillon M. The binding of calcium to bovine fibrinogen. Biochim Biophys Acta 1977; 490: 94-103
• 12 Van Ruijven-Vermeer AM, Nieuwenhuizen W, Nooijen WJ. Calcium binding of rat fibrinogen and fibrin(ogen) degradation products. FEBS Lett 1978; 93: 177-180
• 13 Dang CV, Ebert RF, Bell WR. Localization of a fibrinogen calcium binding site between γ-subunit positions 311 and 336 by terbium fluorescence. J Biol Chem 1985; 260: 9713-9719
• 14 Nieuwenhuizen W, Vermond A, Hermans J. Evidence for the localization of a calcium-binding site in the amino-terminal disulphide knot of fibrin(ogen). Thromb Res 1983; 31: 81-86
• 15 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970; 227: 680-685
• 16 Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 1975; 256: 495-497
• 17 Asakura S, Yoshida N, Matsuda M, Murayama H, Soe G. Preparation and characterization of monoclonal antibodies against the human thrombin-antithrombin complex. Biochim Biophys Acta 1988; 952: 37-47
• 18 Matsuda M, Baba M, Morimoto K, Nakamikawa C. “Fibrinogen Tokyo II”. An abnormal fibrinogen with an impaired polymerization site on the aligned DD domain of fibrin molecules J Clin Invest 1983; 72: 1034-1041
• 19 Frankel ME, Gerhard W. The rapid determination of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus Mol Immunology 1979; 16: 101-106
• 20 Terukina S, Matsuda M, Hirata H, Takeda Y, Miyata T, Takao T, Shimonishi Y. Substitution of γ Arg-275 by Cys in an abnormal fibrinogen “fibrinogen Osaka II”. Evidence for a unique solitary cystine structure at the mutation site J Biol Chem 1988; 633: 13579-13587
• 21 Yamazumi K, Doolittle RF. The synthetic peptide Gly-Pro-Arg-Pro-amide limits the plasmic digestion of fibrinogen in the same fashion as calcium ion. Protein Science 1992; 1: 1719-1720
• 22 Townsend RR, Hilliker E, Li Y-T. Laine RA, Bell WR, Lee YC. Carbohydrate structure of human fibrinogen. Use of 300-MHz¹H-NMR to characterize glycosidase-treated glycopeptides J Biol Chem 1982; 257: 9704-9710
• 23 Dang CV, Shin CK, Bell WR, Nagaswami C, Weisel JW. Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly. J Biol Chem 1989; 264: 15104-15108
• 24 Matsuda M. Abnormal fibrinogens. The present status of structural elucidation Biomed Progr 1991; 4: 51-54
• 25 Ebert RF. Index of Variant Human Fibrinogens. CRC Press; Boca Raton, FL: 1991
• 26 Koopman J, Haverkate F, Grimbergen J, Egbring R, Lord ST. Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids Aa 461-610 (Lys 461 AAA → Stop TAA). Blood 1992; 80: 1972-1979