Biophysical Journal, Volume 82, Issue 4, 1 April 2002, Pages 1835-1847
Eric Y.H. Park, McRae J. Smith, Emily S. Stropp, Karen R. Snapp, Jeffrey A. DiVietro, William F. Walker, David W. Schmidtke, Scott L. Diamond and Michael B. Lawrence
A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophil’s surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21μm at 0.4dyn/cm2, and increased to 1.58μm at 2dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152pN/μm at 0.4 and 2.0dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.
Biophysical Journal, Volume 94, Issue 3, 1 February 2008, Pages 1034-1045
Christopher D. Paschall, William H. Guilford and Michael B. Lawrence
L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14±0.04 bonds/μm at 0.2 dyn/cm2 to 0.014±0.003 bonds/μm at 1.0 dyn/cm2. In contrast, L-selectin tether bond formation increased from 0.017±0.005 bonds/μm at 0.2 dyn/cm2 to 0.031±0.005 bonds/μm at 1.0 dyn/cm2. L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.
P-selectin glycoprotein ligand-1 (PSGL-1) is a large (240 kDa) glycoprotein found on the surface of nearly all leukocytes. The mature molecule is decorated with multiple N- and O-linked glycans and displays copies of the tetrasaccharide sialyl-Lewisx (sLeX), as well as a cluster of three tyrosine sulfate (tyr-SO3) groups near the N-terminus of the processed protein. Previous studies have suggested that PSGL-1 needs to be tyrosine-sulfated, in addition to glycosylated with sLeX, to successfully interact with P-selectin. To better understand how biochemical features of the PSGL-1 ligand are related to its adhesion phenotype, we have measured the dynamics of adhesion under flow of a series of well-defined PSGL-1 variants that differ in their biochemical modification, to both P- and E-selectin-coated substrates. These variants are distinct PSGL-1 peptides: one that possesses sLeX in conjunction with three N-terminal tyr-SO3 groups (SGP3), one that possesses sLeX without tyrosine sulfation (GP1), and one that lacks sLeX but has three N-terminal tyr-SO3 groups (SP3). Although all peptides expressing sLeX, tyr-SO3, or both supported some form of rolling adhesion on P-selectin, only peptides expressing sLeX groups showed rolling adhesion on E-selectin. On P-selectin, the PSGL-1 peptides demonstrated a decreasing strength of adhesion in the following order: SGP3>GP1>SP3. Robust, rolling adhesion on P-selectin was mediated by the GP1 peptide, despite its lack of tyrosine sulfation. However, the addition of tyrosine sulfation to glycosylated peptides (SGP3) creates a super ligand for P-selectin that supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates. Tyrosine sulfation has no similar effect on PSGL-1 rolling on E-selectin. Such functional distinctions in rolling dynamics are uniquely realized with a cell-free system, which permits precise, unambiguous identification of the functional activity of adhesive ligands. These findings are consistent with structural and functional characterizations of the interactions between these peptides and E- and P-selectin published recently.