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

Abstract

SummaryThe 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.

Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration

“…Purified FBG missing the B␤ 1-42 domain, designated FBG-325, 24 was a kind gift from Dr. A. Budzynski (Temple University, Philadelphia, PA). Purified and matrix-incorporated FBG were characterized with the following FBG-specific monoclonal antibodies (MoAbs): anti-B␤ 1-21 (18C6) 25 and anti-␤ 15-21 (T2G1) 26 from Accurate Chemical & Scientific, Westbury, NY; anti-B␤ 262-269 (D73H) 27 and anti-FPA A␣ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] (RDV3), a generous gift from Dr J. R. Shainoff, Cleveland State University, OH; and anti-A␣-RGDF 95-98 (LJ155B16) and anti-A␣-RGDS 572-575 (LJ134B29), generous gifts from Dr Z. Ruggeri, Scripps Research Institute, La Jolla, CA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were performed as previously described.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were performed as previously described. 23,27 Wound repair model Human foreskin fibroblast (HFF) primary cultures were isolated from foreskins obtained from a local hospital as previously described. 28 All tissue collection was approved by the University of Rochester's internal review board for ethical use of discarded human tissues.…”

A putative role for platelet-derived PPARγ in vascular homeostasis demonstrated by anti-PPARγ induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis

“…4A). For production of high titer antibody in ascites fluid, retired breeder mice were injected IP with 1×10 6 hybridoma cells producing the anti-PPARγ-14mer IgM isotype MoAb 4A5 and 3C11 or a well-characterized IgM isotype control MoAb, D73H, specific for the Bβ-chain of human fibrinogen (Rybarczyk et al, 2000). Ascites fluid recovered from the anti-14mer mice (4A5 and 3C11) was unusually bloody.…”

“…Because we observed thrombocytopenia in the anti-PPARγ-14mer rabbits, platelet counts were performed on ascites mice producing MoAb 3C11 (anti-PPARγ-14mer; IgM isotype) and, as an isotype control, D73H (anti-Bβ-chain of human fibrinogen) (Rybarczyk et al, 2000). MoAb D73H was used as a control hybridoma line for ascites production for two reasons.…”

“…Excessive hemorrhaging was observed in the spleens of immunized mice and in the lymph nodes and intraperitoneal tumors of the mice injected with anti-PPARγ hybridoma cells for MoAb production in ascites fluid. Furthermore, platelet counts were substantially reduced in the anti-PPARγ-14mer 3C11 (IgM isotype) ascites mice, but not in the D73H ascites mice producing an IgM isotype MoAb against the Bβ-chain of human fibrinogen (Rybarczyk et al, 2000). Platelet depletion and increased MPV reflect the state of thrombogenesis (Hekimsoy et al, 2004).…”