P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry

Abstract

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P- selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P- selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.

Circulating platelet–neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing

“…Therefore, following ADP stimulation, the entire neutrophil population was found to express more CD11b/CD18, less CD62L, more antibody 24 binding sites, produce more superoxide and phagocytose more N. meningitidis. These results extend the ®ndings from previous studies which showed that platelet activation is a potent stimulus for both PNC formation and neutrophil function (Peters et al, 1997;de Bruijne Admiraal et al, 1992;Chignard & Renesto, 1994;Nagata et al, 1993).…”

“…These ®ndings are indicative that neutrophils within platelet±neutrophil complexes represent an activated subset of cells. In addition, it can be inferred that the platelet component of PNCs must also represent an activated subpopulation of platelets, as CD62P expression is an essential prerequisite for PNC formation (de Bruijne Admiraal et al, 1992;Evangelista et al, 1993).…”

Activation states of blood eosinophils in asthma

“…Eosinophils express seven integrins, a 4 b 1 (CD49d/ CD29), a 6 b 1 (CD49f/CD29), a L b 2 (CD11a/CD18), a M b 2 (CD11b/CD18), a X b 2 (CD11c/CD18), a D b 2 , and a 4 b 7 [5,12,13,41,42,55,128], which potentially interact with ligands including vascular cell adhesion molecule (VCAM)-1, ICAM-1, laminin, fibrinogen/fibrin, vitronectin, and periostin, on other cells or in the extracellular matrix (ECM) [41,42,67]. The platelet integrin a IIb b 3 (CD41/CD61) [147] is not synthesized by eosinophils [110] but can be detected by flow cytometry or immunofluorescence microscopy staining on a variable proportion of eosinophils (in humans and mice), due to association of platelets or platelet fragments with some of the cells [64,130,[148][149][150][151][152]; activated platelets are known to bind leucocytes via P-selectin [153]. Some workers have detected low levels of additional integrins, including a 2 b 1 (CD49b/CD29), which is also not synthesized by eosinophils [110], on the eosinophil surface [154][155][156]; this is similarly likely due to platelet 'satellitism'.…”

Serum‐Soluble Selectin Levels in Patients with Rheumatoid Arthritis and Systemic Sclerosis

“…Upon the activation of these cells by strong agonists, e.g. thrombin, histamine and inflammatory cytokines such as interleukin‐1 and tumour necrosis factor‐α, P‐selectin is expressed on endothelial cells and plays an important role in mediating rolling of leucocytes on the activated endothelial cells of the blood vessel wall in the early phases of inflammatory reactions [10]. Inhibition or loss of one or more selectins significantly attenuates rolling and emigration in several inflammatory models [4].…”