Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: Role of PSGL-1 as a signaling molecule

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is import ant for the recruitment of PMN at sites of vascular damage and thrombus for mation. We have recently shown that binding of activated platelets to PMN i n mixed cell suspensions under shear involves P-selectin and the activated beta(2)-integrin CD11b/CD18. Integrin activation required signaling mechani sms that were sensitive to tyrosine kinase inhibitors.(1) Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under s hear conditions leads to rapid and fully reversible tyrosine phosphorylatio n of a prominent protein of 110 kD (P similar to 110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-select in or CD11b/CD18, suggesting that both adhesion molecules need to engage wi th their respective ligands to trigger phosphorylation of P similar to 110. The inhibition of P similar to 110 phosphorylation by tyrosine kinase inhi bitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin-transfe cted Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P -selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-select in-IgG-triggered PMN/PMN aggregation as well as P similar to 110 phosphoryl ation were all blocked by antibodies against P-selectin or CD18. In each ca se PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein, T he antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering li gand for P-selectin in this experimental system. Moreover, engagement of PS GL-1 with a nonadhesion blocking antibody triggered beta(2)-integrin-depend ent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosin e kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. Th e availability of the Pn-integrin to engage with its ligands on the neighbo ring cells is necessary for the tyrosine phosphorylation of P similar to 11 0. (C) 1999 by The American Society of Hematology.