Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow

Abstract

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin–binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-β-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin–ligand interactions.

Direct observation of catch bonds involving cell-adhesion molecules

“…The tethers were eliminated by coating the floor with human serum albumin instead of (s)P-selectin or by the inclusion of the anti-P-selectin mAb G1, the anti-PSGL-1 mAb PL1 or EDTA in the medium, confirming their specificity. The force f on the tether of a neutrophil or a microsphere was calculated on the basis of the tether angle, which was derived after directly measuring the lever arm of the tether by a flow reversal method 19 . The conversion factors between wall shear stress and tether force for neutrophils and microspheres are 112 and 131 pN dyn 21 cm 22 , respectively.…”

“…3a), approximately doubling the forces and lifetimes. As depicted in Figs 1b and 4a, native membrane P-selectin and PSGL-1 are dimeric, whereas sPSGL-1 is monomeric 12,19,23 . The AFM data suggest that sPSGL-1 forms monomeric bonds with P-selectin, whereas PSGL-1 forms dimeric bonds with P-selectin, which was further supported by molecular elasticity measurements (B.T.M., K. K. Sarangapani, A. Leppänen, R. D. Cummings, R.P.McE.…”

“…We captured dimeric PSGL-1 purified from human neutrophils 18 or monomeric recombinant soluble PSGL-1 (sPSGL-1) 19 with PL2, a non-blocking anti-PSGL-1 mAb 20 adsorbed on the cantilever tip (Fig. 1b).…”

Deformable gas-filled microbubbles targeted to P-selectin

“…Leukocyte deformation correlates with the fluid force acting upon it [18,22]. It has been hypothesized that this deformation enhances a leukocyte's state of adhesion by increasing the leukocyteendothelium contact area, thus augmenting the potential number of adhesive bonds [22,23,24]. The ability to deform may further stabilize adhesion by diminishing the profile of the attached leukocyte in the shear field, thus reducing the dislodging force on the adherent cell [25].…”

Surface-bound selectin–ligand binding is regulated by carrier diffusion

“…For example, contribution of cellular deformability to flowinduced cell adhesion was assumed to play an important role in stable, shear-resistant rolling (Yago et al 2002). Enhancing carrier stiffness and roughing carrier surface reduced effective affinity of receptor-ligand interactions (Williams et al 2001;Wu et al 2007).…”