Characterization of a Monoclonal Antibody, D73H, that Maps to a Highly Conserved Region on Fibrinogen Bβ Chain

 

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Summary

The primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bβ chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bβ chain of plasmin fragment D, localizing its epitope to Bβ134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bβ243-253, further localizing the epitope to Bβ243-305. In silico analysis indicated that Bβ243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bβ252-258, Bβ262-269, and Bβ279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bβ262-269 (FGRKWDPY) is predominantly α-helical and located on the surface of the molecule adjacent to a bend imposed in the β chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bβ261-272 competitively inhibited the binding of D73H to the Bβ chain of denatured intact fibrinogen and reduced and denatured Bβ chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bβ chain residues Bβ261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.

• References

• 1 Odrljin TM, Shainoff JR, Lawrence SO, Simpson-Haidaris PJ. Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin β chain. Blood 1996; 88: 2050-61.
  PubMedGoogle Scholar
• 2 Doolittle RF, Goldbaum DM, Doolittle LR. Designation of sequences involved in the “coiled-coil” interdominal connections in fibrinogen: constructions of an atomic scale model. J Mol Biol 1978; 120: 311-25.
  CrossrefPubMedGoogle Scholar
• 3 Doolittle RF. A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives. Protein Sci 1992; 01: 1563-77.
  CrossrefPubMedGoogle Scholar
• 4 Hantgan RR, Simpson-Haidaris PJ, Francis CW, Marder VJ. Fibrinogen structure and physiology. In: Colman RW, Hirsh J, Marder VJ, Salzman EW. eds. Haemostasis and Thrombosis: Basic Principles and Clinical Practice. 4th ed. Philadelphia: JB Lippincott Company, in press.;
  Google Scholar
• 5 Kudryk B, Rohoza A, Ahadi M, Chin J, Wiebe ME. Specificity of a monoclonal antibody for the NH₂-terminal region of fibrin. Mol Immunol 1984; 21: 89-94.
  CrossrefPubMedGoogle Scholar
• 6 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human but not fibrinogen. Science 1983; 222: 1129-32.
  CrossrefPubMedGoogle Scholar
• 7 Fair DS, Edgington TS, Plow EF. Immunochemical mapping of the conformation of human fibrinogen. The γ95-265 segment in inaccessible to antibody in native fibrinogen but progressively exposed by plasmic cleavage. J Biol Chem 1981; 256: 8018-23.
  PubMedGoogle Scholar
• 8 Kudryk B, Rohoza A, Ahadi M, Chin J, Wiebe ME. A monoclonal antibody with ability to distinguish between NH₂-terminal fragments derived from fibrinogen and fibrin. Mol Immunol 1983; 20: 1191-200.
  CrossrefPubMedGoogle Scholar
• 9 Odrljin TM, Rybarczyk BJ, Francis CW, Lawrence SO, Hamaguchi M, Simpson-Haidaris PJ. Calcium modulates plasmin cleavage of the fibrinogen D fragment γ chain N-terminus: mapping of monoclonal antibody J88B to a plasmin sensitive domain of the γ chain. Biochim Biophys Acta 1996; 1298: 69-77.
  CrossrefPubMedGoogle Scholar
• 10 Haidaris PJ, Francis CW, Sporn LA, Arvan DS, Collichio FA, Marder VJ. Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of γ chain-variant polypeptides. Blood 1989; 74: 743-50.
  PubMedGoogle Scholar
• 11 Koppert PW, Koopman J, Haverkate F, Nieuwenhuizen W. Production and characterization of a monoclonal antibody reactive with a specific neoantigenic determinant (comprising Bβ54-118) in degradation products of fibrin and of fibrinogen. Blood 1986; 68: 437-41.
  PubMedGoogle Scholar
• 12 Haidaris PJ, Peerschke EI, Marder VJ, Francis CW. The C-terminal sequences of the γ57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin. Blood 1989; 74: 2437-44.
  PubMedGoogle Scholar
• 13 Harlow E, Lane D. Antibodies, a laboratory manual Cold Spring Harbor. CSHL Press; 1988
  Google Scholar
• 14 Odrljin TM, Francis CW, Sporn LA, Bunce LA, Marder VJ, Simpson-aidaris PJ. Heparin-binding domain of fibrin mediates its binding to endothelial cells. Arteriosclerosis, Thrombosis Vascular Biol 1996; 16: 1544-51.
  CrossrefPubMedGoogle Scholar
• 15 Lawrence SO, Wright TW, Francis CW, Fay PJ, Haidaris PJ. Purification and functional characterization of homodimeric γB-γ B fibrinogen from rat plasma. Blood 1993; 82: 2406-13.
  PubMedGoogle Scholar
• 16 Simpson-Haidaris PJ, Wright TW, Earnest BJ, Hui Z, Neroni LA, Courtney MA. Cloning and characterization of a lung-specific cDNA corresponding to the γ chain of hepatic fibrinogen. Gene 1995; 167: 273-8.
  CrossrefPubMedGoogle Scholar
• 17 Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 1977; 252: 1102-6.
  PubMedGoogle Scholar
• 18 Everse SJ, Spraggon G, Veerapandian L, Riley M, Doolittle RF. Crystal structure of fragment double-D from human fibrin with two different bound ligands. Biochemistry 1998; 37: 8637-42.
  CrossrefPubMedGoogle Scholar
• 19 Cierniewski CS, Budzynski AZ. Involvement of the α chain in fibrin clot formation. Effect of monoclonal antibodies. Biochemistry 1992; 31: 4248-53.
  CrossrefPubMedGoogle Scholar
• 20 Kini RM, Evans HJ. Prediction of potential protein-protein interaction sites from amino acid sequence. Identification of a fibrin polymerization site. FEBS Lett 1996; 385: 81-6.
  CrossrefPubMedGoogle Scholar
• 21 Kini RM, Evans HJ. A hypothetical structural role for proline residues in the flanking segments of protein-protein interaction sites. Biochem Biophys Res Commun 1995; 212: 1115-24.
  CrossrefPubMedGoogle Scholar
• 22 Chang MC, Jeng JH, Cheong TC, Huang TF. The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by αvβ3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C. Biochem Biophys Acta 1995; 1269: 115-21.
  CrossrefPubMedGoogle Scholar
• 23 Chang MC, Wang BR, Huang TF. Characterization of endothelial cell differential attachment to fibrin and fibrinogen and its inhibition by Arg-Gly-Asp-containing peptides. Thromb Haemost 1995; 74: 764-9.
  Article in Thieme ConnectPubMedGoogle Scholar
• 24 Altieri DC, Plescia J, Plow EF. The structural motif glycine 190-valine 202 of the fibrinogen γ chain interacts with CD11b/CD18 integrin (αMβ2, Mac-1) and promotes leukocyte adhesion. J Biol Chem 1993; 268: 1847-53.
  PubMedGoogle Scholar
• 25 Altieri DC, Duperray A, Plescia J, Thornton GB, Languino LR. Structural recognition of a novel fibrinogen γ chain sequence (117-133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction. J Biol Chem 1995; 270: 696-9.
  CrossrefPubMedGoogle Scholar
• 26 Cheresh DA, Berliner SA, Vicente V, Ruggeri ZM. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell 1989; 58: 945-53.
  CrossrefPubMedGoogle Scholar
• 27 Rudchenko S, Trakht I, Sobel JH. Comparative structural and functional features of the human fibrinogen αC domain and the isolated αC fragment. Characterization using monoclonal antibodies to defined COOH-terminal Aα chain regions. J Biol Chem 1996; 271: 2523-30.
  CrossrefPubMedGoogle Scholar
• 28 Hawiger J. Adhesive interactions of platelets and their blockade. Ann N Y Acad Sci 1991; 614: 270-8.
  CrossrefPubMedGoogle Scholar
• 29 Francis CW, Bunce LA, Sporn LA. Endothelial cell responses to fibrin mediated by FPB cleavage and the amino terminus of the β chain. Blood Cells 1993; 19: 291-306.
  PubMedGoogle Scholar
• 30 Bunce LA, Sporn LA, Francis CW. Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15–42 of the β chain. J Clin Invest 1992; 89: 842-50.
  CrossrefPubMedGoogle Scholar
• 31 Hamaguchi M, Bunce LA, Sporn LA, Francis CW. Spreading of platelets on fibrin is mediated by the amino terminus of the β chain including peptide β15-42. Blood 1993; 81: 2348-56.
  PubMedGoogle Scholar
• 32 Sporn LA, Bunce LA, Francis CW. Cell proliferation on fibrin: modulation by fibrinopeptide cleavage. Blood 1995; 86: 1802-10.
  PubMedGoogle Scholar
• 33 Francis CW, Marder VJ. A molecular model of plasmic degradation of crosslinked fibrin. Sem Thromb Hemost 1982; 08: 25-35.
  Article in Thieme ConnectPubMedGoogle Scholar
• 34 Budzynski AZ, Marder VJ. Degradation pathway of fibrinogen by plasmin. Thromb Haemost 1977; 38: 793-800.
  Article in Thieme ConnectPubMedGoogle Scholar
• 35 Bacon-Baguley T, Ogilvie ML, Gartner TK, Walz DA. Thrombospondin binding to specific sequences within the Aα-and Bβ-chains of fibrinogen. J Biol Chem 1990; 265: 2317-23.
  PubMedGoogle Scholar
• 36 Tran H, Tanaka A, Litvinovich SV, Medved LV, Haudenschild CC, Argraves WS. The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis. J Biol Chem 1995; 270: 19458-64.
  CrossrefPubMedGoogle Scholar
• 37 Godyna S, Diaz-Ricart M, Argraves WS. Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen. Blood 1996; 88: 2569-77.
  PubMedGoogle Scholar
• 38 Guadiz G, Sporn LA, Simpson-Haidaris PJ. Thrombin cleavage-independent deposition of fibrinogen in extracellular matrices. Blood 1997; 90: 2644-53.
  PubMedGoogle Scholar