Neutrophil expression of CD41/CD61 complex contributes to their adhesiveness in both healthy individuals and heart failure patients

Abstract

Background: Neutrophils can release pro-inflammatory cytokines and neutrophil extracellular traps (NETs), leading to vascular thrombosis. Neutrophil-platelet interaction, a major component of thrombosis, is more pronounced in inflammatory pathologies, such as heart failure (HF). Recently, the platelet receptor CD41/CD61 (GPIIb/IIIa), known for its role in platelet adhesion and aggregation via its binding to fibrinogen, was found on the membrane of neutrophils from lung cancer patients. Our objectives were to determine CD41/CD61 expression on neutrophils and its role in healthy volunteers (HV) and acute decompensated heart failure patients (ADHF).

Results: Localization and expression of CD41/CD61 complex on isolated neutrophils were determined by flow cytometry and confocal microscopy. We assessed the CD41/CD61 role on neutrophil adhesion onto human extracellular matrix (hECM), NETosis and release of inflammatory cytokines. CD41/CD61 complex was intracellularly expressed in 80–90% of neutrophils, but only between 8 and 13% on their extracellular membrane. The CD41/CD61 complex plays a role in neutrophil adhesion onto hECM, since its blockade by an anti-CD41 monoclonal antibody or its antagonist, eptifibatide, reduced CXCL8 and PMA induced neutrophil adhesion from 52 to 100% in both HV and ADHF patients.

Conclusions: In summary, neutrophil specific CD41/CD61 expression contributes to neutrophil adhesiveness without affecting significantly the release of inflammatory cytokines and NETosis in both HV and ADHF patients.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12865-025-00742-3.

Keywords: Adhesion; Heart failure; NETosis; Neutrophils.

Fig. 1

Flow cytometry detection of CD41/CD61 complex on isolated neutrophils. Purity of post-isolation neutrophils was analyzed by flow cytometry using the forward scatter (FSC) and side scatter (SSC) (A-B) then neutrophils were stained with a CD66b fluorescent antibody (mouse anti-human CD66b-AF647) or its isotype control (mouse IgM) (C). Non-permeabilized (NP) or permeabilized (P) neutrophils isolated from HV (NP; n = 28, P; n = 16–19) or ADHF (NP; n = 21–22, P; n = 8–11) were incubated with a CD41/CD61 fluorescent antibody (mouse anti-human CD41/CD61-FITC). An example of flow cytometry data is shown for each condition in panel B. The percentage of neutrophils expressing CD41/CD61 was calculated using an IgG-control antibody (C). The mean fluorescence intensity (MFI) ratio was determined by dividing the MFI of CD41/CD61 Ab by its corresponding IgG (D). ****p < 0.0001 vs. expression of CD41/CD61 complex on non-permeabilized neutrophils

Fig. 2

Representative expression of CD41/CD61 complex by confocal microscopy on neutrophils from a HV. Neutrophils were incubated with PMA (25 nM; 3 h) then stained with a CD41/CD61 marker (mouse anti-human CD41/CD61-FITC; green), a neutrophil marker (mouse anti-human CD66b-Alexa Fluor 647; red), and a nuclear marker (Hoechst 33342; blue). Confocal microscopy pictures were taken at a magnification of 630X (LSM 710, Carl Zeiss). Staining was performed on non-permeabilized and permeabilized neutrophils

Fig. 3

Time-dependent flow cytometry detection of CD41/CD61 complex on isolated neutrophils. Non-permeabilized neutrophils isolated from HV (n = 3–6) were incubated with either a CD41/CD61 fluorescent antibody (mouse anti-human CD41/CD61-FITC) or its control IgG antibody, then stimulated with PBS, PMA (25 nM) or CXCL8 (100 nM) for 0, 7.5, 15, 30 and 60 min. The percentage of neutrophils expressing CD41/CD61 was calculated using the IgG-control antibody. *p < 0.05 vs. PBS at corresponding time and †P < 0.05 vs. CXCL8 at 0 min.

Fig. 4

Isolated neutrophil adhesion onto hECM. Isolated neutrophils from (A) HV (n = 9–22) and (B) ADHF patients (n = 9–16) were pre-treated with PBS, DMSO (Additional File 1), GW311616, anti-human CD41 Ab and/or ß2 integrin/CD18 Ab and their respective IgGs (Additional File 1) or eptifibatide for 30 min, then incubated with PBS, PMA (25 nM) and CXCL8 (100 nM) for an additional 7.5 min on hECM coated 48-well plates. Adhered neutrophils were counted in 4 fields of view per well by optical microscopy using a digital camera. Adhesion of neutrophils was represented as the average number of adhered neutrophils / field from the four FOV of each well. C and D are comparative layouts of the same data presented in panels A and B. All values are presented as mean ± SEM. P < 0.05 was considered statistically significant. (∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001 vs. corresponding PBS-treated neutrophils; †††P < 0.001 and ††††P < 0.0001 vs. corresponding control pre-treatment)

Fig. 5

NETosis in isolated neutrophils from HV and ADHF patients. Isolated neutrophils from (A) HV (n = 5) and (B) ADHF patients (n = 5) were pre-treated with PBS, DMSO, GW-311616 and eptifibatide for 30 min prior to incubation with PBS, PMA (25 nM) and CXCL8 (100 nM; Additional File 1) for an additional 3 h in 24-well plates. NETs were isolated by nuclease S7 DNA digestion and quantified by a MPO-DNA custom ELISA. All values are presented as mean ± SEM. P < 0.05 was considered statistically significant. (∗∗P < 0.01 and ∗∗∗P < 0.001 vs. corresponding PBS-treated neutrophils; †P < 0.05 and ††††P < 0.0001 vs. DMSO)

Fig. 6

Inflammatory release in isolated neutrophils from HV and ADHF patients. Isolated neutrophils from (A) HV (n = 4–5) and (B) ADHF patients (n = 4–5) were pre-treated with PBS, DMSO, GW-311616 and eptifibatide for 30 min, then treated with PBS, PMA (25 nM) and CXCL8 (100 nM; Additional File 1) for an additional 3 h in 24-well plates. Cellular supernatant was collected for CXCL8, IL-1RA and VEGF measurements by multiplex assay. All values are presented as mean ± SEM. P < 0.05 was considered statistically significant. (∗P < 0.05, ∗∗P < 0.01 and ∗∗∗∗P < 0.0001 vs. corresponding PBS-treated neutrophils; †P < 0.05 vs. DMSO)