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

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.