Residues F16-G33 and A784-N823 within platelet thrombospondin-1 play a major role in binding human neutrophils: evaluation by two novel binding assays

J Lab Clin Med. 2000 Oct;136(4):292-302. doi: 10.1067/mlc.2000.109407.

Abstract

The thrombospondin-1 (TSP1) structural requirements within its heparin-binding domain (HBD)(30 kd) or within the other domains of the molecule (450 kd) that interact with neutrophils (PMNs) have not been delineated. Synthetic peptides based on the HBD, a TSP1 proteolytic fragment lacking the HBD, a large C-terminal domain of TSP1 (210 kd), a TSP1 recombinant fragment (rTSP1(784-932)), and a monoclonal antibody directed against the TSP1 type 3 repeats (mAb D4.6) were utilized to map such structural requirements on TSP1. Synthetic peptides containing a heparin-binding motif and encompassing residues F16-G33 or A74-S95 of TSP1 competed quantitatively with iodine 125-labeled TSP1 for binding to heparinagarose beads. However, only F16-G33 was a competitor of TSP1 binding to PMNs, suggesting that the sequence F16-G33 within the HBD plays a role in PMN binding. The interaction site within the 450-kd fragment was further narrowed. A TSP1 -derived proteolytic fragment (210 kd), a recombinant TSP1 fragment (rTSP1(784-932)), and a type 3 repeat anti-TSP1 monoclonal antibody (mAb D4.6) competed for the binding of 125I-labeled TSP1 to PMNs. The N-terminal of rTSP1(784-932) and C-terminal sequence analysis of TSP1-210 kd delineated the structural requirements for the second binding region for PMNs-namely, residues A784-N823.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal
  • Binding Sites / drug effects
  • Binding Sites / immunology
  • Blood Platelets / chemistry
  • Blood Platelets / metabolism*
  • Calcium / metabolism
  • Chemotaxis, Leukocyte / drug effects
  • Chemotaxis, Leukocyte / immunology
  • Fibrinolytic Agents / metabolism
  • Fibrinolytic Agents / pharmacology
  • Fluorescein-5-isothiocyanate
  • Fluorescent Dyes
  • Heparin / metabolism
  • Heparin / pharmacology
  • Humans
  • Iodine Radioisotopes
  • Molecular Sequence Data
  • Neutrophils / cytology
  • Neutrophils / metabolism*
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / pharmacology
  • Peptide Mapping
  • Protein Structure, Tertiary
  • Sensitivity and Specificity
  • Thrombospondin 1 / chemistry
  • Thrombospondin 1 / immunology
  • Thrombospondin 1 / metabolism*

Substances

  • Antibodies, Monoclonal
  • Fibrinolytic Agents
  • Fluorescent Dyes
  • Iodine Radioisotopes
  • Peptide Fragments
  • Thrombospondin 1
  • Heparin
  • Fluorescein-5-isothiocyanate
  • Calcium