Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule

Abstract

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected 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-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.

Neutrophils mediate edema formation but not mechanical allodynia during zymosan-induced inflammation

“…It is thought that initial tethering of the cells is mediated by interaction of platelet P-selectin (CD62P) and neutrophil PSGL-1 (CD162). This is followed by firm adhesion through binding of the ␤ 2 integrin macrophage-1 antigen (CD11b/CD18) on neutrophils to several platelet counter-receptors (GPIb␣, junctional adhesion molecule-C, and fibrinogen bound to GPIIb/IIIa) [55][56][57][58]. It should be noted that several of these receptors are also involved in the interaction of both cell types to endothelial cells.…”

Platelets: key players in vascular inflammation

“…This binding may occur in circulation or upon the adherence of platelets to the vascular wall, thereby providing an adherent surface to recruit leukocytes [3,41]. Leukocytes initially adhere to platelets via PSGL-1-P-selectin interactions [42,43] and this adhesion is subsequently stabilized through binding of Mac-1 (CD11b/CD18, ␣ M ␤ 2 ) to the GPIb␣ [44]. Engagement of PSGL-1 and Mac-1 induces an inflammatory cascade in monocytes, causing them to secrete effector molecules such as CCL5 [45,46].…”

Severe meningococcal disease is characterized by early neutrophil but not platelet activation and increased formation and consumption of platelet–neutrophil complexes

“…The resulting plateletneutrophil complexes (PNC) are formed by a multistep adhesion process [1] initiated by platelet CD62P-mediated adhesion to PMNL P-selectin glycoprotein ligand-1. This initial adhesion induces CD11b/CD18 (Mac-1) activation [2] via SRC kinases, which then firmly adhere to a second platelet ligand, probably fibrinogen bound to the activated form of the platelet integrin ␣IIb/␤3 (GpIIb/IIIa). Other molecules such as CD40 and CD40L found on the platelet also act to link the haemostatic and inflammatory pathways [3].…”