Under physiological shear stress, neutrophils roll on P-selectin on activated endothelial cells or platelets through interactions with P-selectin glycoprotein ligand-1 (PSGL-1). Both P-selectin and PSGL-1 are extended molecules. Human P-selectin contains an NH2-terminal lectin domain, an EGF domain, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the length of P-selectin affected its interactions with PSGL-1, we examined the adhesion of neutrophils to CHO cells expressing membrane-anchored P-selectin constructs in which various numbers of CRs were deleted. Under static conditions, neutrophils attached equivalently to wild-type P-selectin and to constructs containing from 2-6 CRs. Under shear stress, neutrophils attached equivalently to wild-type and 6 CR P-selectin and nearly as well to 5 CR P-selectin. However, fewer neutrophils attached to the 4 CR construct, and those that did attach rolled faster and were more readily detached by increasing shear stress. Flowing neutrophils failed to attach to the 3 CR and 2 CR constructs. Neutrophils attached and rolled more efficiently on 4 CR P-selectin expressed on glycosylation-defective Lec8 CHO cells, which have less glycocalyx. We conclude that P-selectin must project its lectin domain well above the membrane to mediate optimal attachment of neutrophils under shear forces. The length of P-selectin may: (a) facilitate interactions with PSGL-1 on flowing neutrophils, and (b) increase the intermembrane distance where specific bonds form, minimizing contacts between the glycocalyces that result in cell-cell repulsion.
P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils.
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K D Patel, M U Nollert, R P McEver; P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils.. J Cell Biol 15 December 1995; 131 (6): 1893–1902. doi: https://doi.org/10.1083/jcb.131.6.1893
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