Location of peptide fragments in the fibrinogen molecule by immunoelectron microscopy.

Abstract

Antibodies to the disulfide knot fragment of bovine fibrinogen have been used to locate the site of this fragment within the intact fibrinogen molecule. The antibodies were isolated from rabbit antifibrinogen antisera by affinity chromatography. Electron micrographs of reaction mixtures of bovine fibrinogen and antibodies against the disulfide knot fragment showed pairs of fibrinogen molecules crosslinked by antibody molecules as well as higher order antibody-fibrinogen complexes. From an electron microscopic investigation of the crosslinked material, we conclude that the disulfide knot lies within the central nodule of the trinodular fibrinogen molecule. Antibodies to fragment H were used in the same manner to locate this fragment within the outer nodules of the human fibrinogen molecule.In connection with our studies of the interaction of thrombin with fibrinogen (1, 2) and of the polymerization of fibrin monomer (3, 4), it is important to locate the sites of the fibrinogen molecule that are involved in various functions such as thrombin-binding, polymerization, fibrin stabilization, and plasmin degradation. From a series of physicochemical (5, 6) and electron microscopic (4, 7-10) observations, the linear trinodular structure of fibrinogen appears established. In addition, electron microscope observations of dimers, trimers, tetramers, etc. of fibrin monomer (4) have verified the conclusions from earlier physicochemical studies (11) that these intermediate polymers arise by a lateral association with partial overlapping, giving two parallel end-to-end chains with staggered junctions. In this paper, we use the technique of immunoelectron microscopy (12-16) to locate two peptide fragments within the trinodular structure of fibrinogen. These two fragments are the "disulfide knot" (N-DSK) (17) and fragment H (18). The former is the disulfide-linked NH2-terminal portions of the Aa, Bf3, and -y chains (Mr -58,000) produced by CNBr cleavage of fibrinogen (17), and fragment H (Mr 20,000) is a plasmin-digestion product of fibrinogen (18). By comparison of the NH2-terminal sequence of fragment H given by Harfenist and Canfield (18) with the amino acid sequence reported by Doolittle et al. (19), it appears that fragment H corresponds to residues 240-424 of the Aa chain. MATERIALS AND METHODSBovine fibrinogen (96% clottable) was prepared from Miles fraction I (lot 90, 75% clottable) or Sigma fraction I (lot 117C-0140, 82% clottable) as described (20) and was dialyzed against 10 mM Na2HPO4/3 mM KH2PO4/120 mM NaCl, pH 7.5 (phosphate-buffered saline), 5 mM H3B04/1.25 mM Na2B4O7.10H20/140 mM NaCl, pH 8.5 (borate/saline buffer), or 50 mM ammonium formate (pH 8.5) for immunization, quantitative precipitation, or electron microscopy, respectively. The reduced and alkylated material exhibited three bands (corresponding to the Aa, BB, and y chains) on NaDodSO4/ 7.5% polyacrylamide gel electrophoresis (21). All other chemicals were of reagent grade or better.Human fibrinogen was purchased from AB Kabi (Stockholm, lot 1981-...

Immune labeling of the D and E regions of human fibrinogen by electron microscopy.

“…Although the majority of workers in the field have accepted the view that the D and E fragments represent the cleaved nodules of the trinodular model, direct evidence on this point has been lacking. Telford et al (31) related the amino-terminal disulfide knot and plasmin fragment H with the central and outer regions of the trinodular structure, respectively. The work described here brings various biochemical results relating to the placement of the D and E regions into closer alignment with the actual physical structure.…”

Fibrinogen and fibrin structure and fibrin formation measured by using magnetic orientation.

“…The trinodular elongated (450-A-long) structure for the fibrinogen molecule proposed by Hall and Slayter (3) is the most widely accepted model, and it has obtained additional support from recent work on native fibrinogen (4)(5)(6)(7)(8) or slightly modified fibrinogen (9)(10)(11). Fibrin monomers are produced by thrombin, which releases the small negatively charged fibrinopeptides A and B.…”

Protein structure and function, from a colloidal to a molecular view

“…It has also been used for peptide synthesis with proteolytic enzymes (26). Limited proteolytic reactions are used to map the active sites ofproteolytic enzymes (295,310,373). For example, with the aid ofa SchechterBerger type of analysis of the kinetics of enzyme action (295), interactions (such as an Arg... Asp salt link in the substrate) involved in the thrombin-fibrinogen complex are being identified ( 18 l, 310).…”