Platelet-neutrophil complex formation—a detailed in vitro analysis of murine and human blood samples

Abstract

Platelets form complexes with neutrophils during inflammatory processes. These aggregates migrate into affected tissues and also circulate within the organism. Several studies have evaluated platelet-neutrophil complexes as a marker of cardiovascular diseases in human and mouse. Although multiple publications have reported platelet-neutrophil complex counts, we noticed that different methods were used to analyze platelet-neutrophil complex formation, resulting in significant differences, even in baseline values. We established a protocol for platelet-neutrophil complex measurement with flow cytometry in murine and human whole blood samples. In vitro platelet-neutrophil complex formation was stimulated with ADP or PMA. We tested the effect of different sample preparation steps and cytometer settings on platelet-neutrophil complex detection and noticed false-positive counts with increasing acquisition speed. Platelet-neutrophil complex formation depends on platelet P-selectin expression, and antibody blocking of P-selectin consequently prevented ADP-induced platelet-neutrophil complex formation. These findings may help generating more comparable data among different research groups that examine platelet-neutrophil complexes as a marker for cardiovascular disease and novel therapeutic interventions.

Intercellular Interactions as Regulators of NETosis

“…Examples of ligand/receptor pairs that mediate direct platelet/neutrophil interactions include P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) (36, 37), intercellular adhesion molecule 2 (ICAM-2)/lymphocyte function-associated antigen (LFA-1) (38), and GPIb/macrophage-1 antigen (Mac-1) (17, 39). These interactions clearly support platelet adhesion to leukocytes (40, 41) and, in some cases, have been shown to be of fundamental importance for recruitment of neutrophils to sites of inflammatory insult (40). Furthermore, beyond traditional direct interaction, some molecules (such as GPIIb/IIIa) may be transferred from platelets to neutrophils via microparticles (MP), thereby regulating neutrophil function (an example being nuclear factor kappa B activation) (42).…”

“…in both perpetuating platelet–neutrophil interplay and activating neutrophils. As an example, ADP (which would presumably be platelet-derived in vivo ) induces platelet–neutrophil complexes through a mechanism that may be dependent upon P-selectin, but not PSGL-1 (41). TXA 2 augments multiple neutrophil functions, including neutrophil adhesiveness (43), oxidative burst (44), and diapedesis (45).…”

Pneumolysin Mediates Platelet Activation In Vitro

“…This study has focused on CD62P because upregulation of expression of this adhesion molecule on platelets is increasingly recognized as being a key mediator of inflammatory events, directing neutrophil trafficking and activation [15]. More recently, CD62P has also been reported to be the primary mediator of heterotypic platelet-neutrophil aggregation [19], a central event in the pathogenesis of arterial thrombogenesis [22]. The mechanism of Ply-mediated platelet activation appears to involve sublytic pore formation and influx of Ca 2+ , resulting in mobilization of CD62P-expressing α-granules.…”

“…In this context, CD62P is widely recognized as being the major platelet-derived mediator of homotypic as well as heterotypic aggregation, the latter involving neutrophils and endothelial cells, activities which are both pro-inflammatory and pro-thrombotic [16,19].…”

Activated CD8 T cells express two distinct P-Selectin Ligands

“…Activated platelets express high densities of PSel (72) and represent a likely, physiologically potent, target of engagement for Tact bearing PSGL1 and LSel/PSL2. Such interactions might occur in circulation (73) and when either platelets or Tact are tethered to endothelia. Platelet--leukocyte interactions can promote recruitment (74--77) and support chemokinetic responses (78).…”