Binding and processing of fibrinogen by rabbit hepatocytes

• Published in: The Journal of biological chemistry Vol. 262; no. 8; pp. 3674 - 3679
• Main Authors: Barsigian, C., Fellin, F.M., Base, W., Schaeffer, A., Fish, S., Martinez, J.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.03.1987; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Binding, Competitive; Biological and medical sciences; fibrinogen; Fibrinogen - metabolism; Fundamental and applied biological sciences. Psychology; Glycoproteins; hepatocytes; In Vitro Techniques; Kinetics; Liver - metabolism; Oligopeptides - pharmacology; Platelet Membrane Glycoproteins - drug effects; Platelet Membrane Glycoproteins - metabolism; Protein Binding; Proteins; Rabbits; Vertebrata; Mammalia; Fixation; Hepatocyte; Animal; Fibrinogen; Rabbit; Lagomorpha; Serum protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)61407-3

We describe a specific fibrinogen-hepatocyte interaction. Rabbit 125I-labeled fibrinogen (125I-FGN) was incubated at 4 degrees C with suspensions of rabbit hepatocytes (approximately 1 × 10(6) cells/ml). Bound ligand was separated from free by centrifugation of cells through oil and quantitated by gamma-scintillation counting. Specific binding, determined by subtraction of nonspecific binding in the presence of 8 mM EDTA from total binding in the presence of 2 mM CaCl2, required 3 h to plateau and represented approximately 70% of total binding. Specific binding was calcium-dependent and was negligible in buffer containing 2 mM MgCl2. Half-maximal saturation occurred at approximately 30 nM 125I-FGN with approximately 480,000 molecules/cell at saturation. Dilution experiments revealed comparable affinities for labeled and unlabeled fibrinogen. Total binding was irreversible as determined by addition of excess unlabeled fibrinogen or EDTA. Specific binding of 25 nM 125I-FGN was inhibited, in a concentration-dependent fashion, by unlabeled fibrinogen or fibrinogen fragment D95 (Mr = 95,000), but not by fibrinogen fragment E or Arg-Gly-Asp-containing peptides. Unlabeled fibrinogen (3.1 microM) completely abolished specific binding, whereas greater than 80% inhibition was achieved with 10 microM fragment D95. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of 125I-FGN bound in the presence of calcium demonstrated disappearance of A alpha chains with formation of products of Mr greater than 200,000; EDTA or unlabeled fibrinogen prevented fibrinogen processing. These data describe a unique fibrinogen-hepatocyte interaction which differs considerably from the platelet-fibrinogen interaction, especially with regard to the processing of the fibrinogen molecule.