Characterization of a mode of specific binding of fibrin monomer through its amino-terminal domain by macrophages and macrophage cell-lines.
The studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (alpha-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX congruent 200-800 molecules/cell, KD congruent to 10(-12) M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX greater than or equal to 10(5) molecules/cell, KD greater than or equal to 10(-6) M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD congruent to 10(-10) M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (beta-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the alpha- and the beta-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with electrophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.
Shainoff
J R
JR
Research Institute, Cleveland Clinic Foundation, OH 44195.
Stearns
D J
DJ
DiBello
P M
PM
Hishikawa-Itoh
Y
Y
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D005337:Fibrin / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008264:Macrophages / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism; D018000:Receptors, Peptide
D000345:Affinity Labels; D005338:Fibrin Fibrinogen Degradation Products; D007155:Immunologic Factors; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C035637:fibrin receptor; D005337:Fibrin