Single molecule characterization of P-selectin/ligand binding

William Hanley, Owen McCarty, Sameer Jadhav, Yiider Tseng, Denis Wirtz, Konstantinos Konstantopoulos

Research output: Contribution to journalArticle

160 Scopus citations

Abstract

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-Selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s-1. The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s-1. Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.

Original languageEnglish (US)
Pages (from-to)10556-10561
Number of pages6
JournalJournal of Biological Chemistry
Volume278
Issue number12
DOIs
StatePublished - Mar 21 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Hanley, W., McCarty, O., Jadhav, S., Tseng, Y., Wirtz, D., & Konstantopoulos, K. (2003). Single molecule characterization of P-selectin/ligand binding. Journal of Biological Chemistry, 278(12), 10556-10561. https://doi.org/10.1074/jbc.M213233200