Monocyte-Platelet Aggregates Triggered by CD31 Molecule in Non-ST Elevation Myocardial Infarction: Clinical Implications in Plaque Rupture

Abstract

Despite the recent innovations in cardiovascular care, atherothrombosis is still a major complication of acute coronary syndromes (ACS). We evaluated the involvement of the CD31 molecule in thrombotic risk through the formation of monocyte-platelet (Mo-Plt) aggregates in patients with ACS with no-ST-segment elevation myocardial infarction (NSTEMI) on top of dual anti-platelet therapy (DAPT). We enrolled 19 control (CTRL) subjects, 46 stable angina (SA), and 86 patients with NSTEMI, of which, 16 with Intact Fibrous Cap (IFC) and 19 with Ruptured Fibrous Cap (RFC) as assessed by the Optical Coherence Tomography (OCT). The expression of CD31 on monocytes and platelets was measured. Following the coronary angiography, 52 NSTEMIs were further stratified according to thrombus grade (TG) evaluation. Finally, a series of ex vivo experiments verified whether the CD31 participates in Mo-Plt aggregate formation. In patients with NSTEMI, CD31 was reduced on monocytes and was increased on platelets, especially in NSTEMI presented with RFC plaques compared to those with IFC lesions, and in patients with high TG compared to those with zero/low TG. Ex vivo experiments documented an increase in Mo-Plt aggregates among NSTEMI, which significantly decreased after the CD31 ligation, particularly in patients with RFC plaques. In NSTEMI, CD31 participates in Mo-Plt aggregate formation in spite of optimal therapy and DAPT, suggesting the existence of alternative thrombotic pathways, as predominantly displayed in patients with RFC.

Keywords: CD31; acute coronary syndromes; monocyte-platelet aggregates; plaque rupture; precision medicine; thrombus burden; unstable plaque.

Figure 1

Flow chart showing the enrolled study population and its allocation within the main experimental procedures. Of 86 patients with NSTEMI, 35 underwent OCT investigation, defining n = 16 IFC, and n = 19 RFC. Obtained PBMC from 80 NSTEMI, 27 patients with SA and 19 CTRL subjects enrolled were evaluated for CD31 expression by flow cytometry. Platelet CD31 was assessed by flow-cytometry on 53 NSTEMI, 36 patients with SA, and 8 CTRL subjects. Thrombus grade analysis was performed on the latter 53 patients with NSTEMI. For co-culture binding experiments, 20 NSTEMI and 6 SA were employed. Finally, 11 were engaged for CD31 ligation experiments; of those patients, 5 were investigated through confocal microscopy. CTRL, control subjects; IFC, intact fibrous cap; NSTEMI, non-ST-elevation myocardial infarction; PBMCs, peripheral blood mononuclear cells; PLTs, platelets; RFC, ruptured fibrous cap; and SA, stable angina patients (art images from http://smart.servier.com/ site).

Figure 2

CD31 expression on monocytes. Graphs showing the surface protein expression of CD31 on CD14⁺ monocytes assessed by flow-cytometry (mean ± SD) in basal conditions. (A) Representative flow cytometry forward vs. side scatter plot of monocyte subset; (B) Representative flow cytometry overlay plot; (C) Data analysis of _moCD31 surface protein expression between the three groups (CTRL, Control individuals; FS, Forward Scatter; MFI, Median Fluorescence Intensity; _moCD31, monocyte CD31; NSTEMI, Non-ST-Elevation Myocardial Infarction; SA, Patients with Stable Angina; SD, Standard Deviation; and SS, Side Scatter).

Figure 3

CD31 expression on platelets. Graphs showing the surface protein expression of CD31 on CD42b⁺ platelets assessed by flow-cytometry (mean ± SD), in basal conditions. (A) Representative flow cytometry forward vs side scatter plot of platelets; (B) Representative flow cytometry overlay plot; (C) Data analysis of pltCD31 surface protein expression between the three groups (CTRL, Control individuals; FS, Forward Scatter; MFI, Median Fluorescence Intensity; NSTEMI, Non-ST-Elevation Myocardial Infarction; pltCD31, platelet CD31; SA, Patients with Stable Angina; SD, Standard Deviation; and SS, Side Scatter).

Figure 4

CD31 expression on monocytes and platelets according to OCT investigation. Dot plots (mean ± SD) and related representative overlay histograms showing the surface protein expression of CD31 within the NSTEMI group that underwent an OCT investigation on CD14⁺ monocytes (A), and CD42b⁺ platelets assessed by flow cytometry (B), in basal conditions. (NSTEMI, Non-ST-Elevation Myocardial Infarction; _moCD31, monocyte CD31; _pltCD31, platelet; RFC, Ruptured Fibrous Cap; IFC, Intact Fibrous Cap; and MFI, Median Fluorescence Intensity.

Figure 5

Platelet CD31 expression and Thrombus-Grade evaluation. (A) Representative angiographic and OCT images of a plaque. (A) Coronary angiogram of the left coronary artery showing a culprit lesion on the left circumflex (dotted squared selection); (B) Magnification of the angiogram showing a complex culprit lesion with a thrombus grade 3 and a scalloped profile (Ambrose type II eccentric); (C) Corresponding OCT cross-sectional image showing a lipid-rich plaque with a disrupted fibrous cap (yellow arrow); (D,E) Adjacent OCT cross-sections showing a large thrombus (yellow arrows). (B) pltCD31 surface protein expression within NSTEMI group showing the differences between patients with zero/low and high thrombus grade at the site of culprit stenosis. Data were assessed by flow-cytometry (mean ± SD), in basal conditions (MFI, Median Fluorescence Intensity; _pltCD31, platelet CD31; SD, Standard Deviation; and TG, Thrombus Grade).

Figure 6

Monocyte-platelet binding. Graphs showing monocyte-platelet (Mo-Plt) binding assessed by flow-cytometry in patients with NSTEMI as compared with patients with SA (A); in patients with NSTEMI before and during the CD31 ligation (B), also according to an OCT investigation (C). Data are presented as % of CD14/CD42b-positive cells (mean ± SD). (NSTEMI, Non-ST-Elevation Myocardial Infarction; SA, Stable Angina; SD, Standard Deviation; and w/, with).

Figure 7

Monocyte-platelet binding at confocal microscopy. Immunofluorescence confocal microscopy images (512 × 512 pixels of resolution, scale bar 20 μm) showing monocyte-platelet (Mo-Plt) binding before and after the CD31 ligation. (A) A representative patient with NSTEMI; (B) 3D plots showing display the changing in Mo-Plt binding after the CD31 ligation; (C) Mo-Plt co-localization within NSTEMI group (n = 5). The degree of co-localization was quantified by using a Mander's overlap coefficient (MOC) (μm = micrometer; NSTEMI, Non-ST-Elevation Myocardial Infarction; w/, with, w/o, without).

Figure 8

CD31 and the thromboinflammatory response of the unstable plaque: A plaque rupture paradigm. The figure summarizes the driving hypothesis that derives from both the existing evidence on plaque rupture and the data that emerged from the present study. In ACS, CD31 is involved in leukocytes and platelets adhesion on ECs. The increased monocyte-platelet (Mo-Plt) binding in the RFC milieu might account for an increased athero-thrombotic burden driven by the inflammation and involving, at least in part, CD31 molecule, as demonstrated by the significant reduction of Mo-Plt aggregate formation following the CD31 ligation (ACS, acute coronary syndromes; and endothelial cells, ECs).