Table 1.
Demographic, clinical and hematological details of all controls and patients participating in the study.
Table 2.
Primer sequences for performance of real-time quantitative PCR.
Figure 1.
Platelets as inflammatory cells in SCA.
Release of (A) IL-1β, (B) IL-8, (C) platelet factor 4 (PF4) from platelets of healthy control individuals (CON) and SCA patients in steady state (SCA). Cytokine release was determined by ELISA in platelet suspensions (1×108 PLT/ml) after incubation for 4 h (37°C, 5% CO2). Expressions of (D) P-selectin and (E) activated αIIbβ3 integrin on the surface of platelets from healthy control individuals (CON) and SCA patients in steady state (SCA). Medians and interquartile ranges are depicted. Adhesion molecule expression was determined by flow cytometry. Some of the data included in the data sets depicted in graphs 1D and 1E have been previously published in table form [14]. Closed black square symbols represent SCA patients not on HU therapy; open grey square symbols represent patients on HU (15–30 mg/kg/day) therapy.
Figure 2.
Adhesive properties of platelets.
A microfluidic assay was used to determine the adhesion of platelets from healthy control individuals (CON) and SCA patients in steady state (SCA) to (A) 50 µg/ml fibrinogen and (B) 100 µg/ml collagen. Platelets (1×107 PLTs/mL) were perfused over microchannels (400 µm width) coated with immobilized ligands at a shear stress of 0.3 dyne/cm2 for 3 min at 37°C; adhesion was detected using brightfield inverted microscopy. The adhesion of platelets to duplicate microchannels was recorded at three positions in each channel and analyzed using the DucoCell analysis program, recording the mean number of platelets adhered to an area of 0.08 mm2. Medians and interquartile ranges are depicted. Closed black square symbols represent SCA patients not on HU therapy; open grey square symbols represent patients on HU (15–30 mg/kg/day) therapy.
Figure 3.
Effects of platelets on endothelial adhesion molecule expression.
Surface expressions of (A) ICAM-1 (CD54), (B) E-selectin (CD62E) and (C) VCAM-1 (CD106) adhesion molecules on HUVEC following co-culture in direct contact with healthy control (CON; N≥15) PLTs, steady-state sickle cell anemia (SCA; N≥25) PLTs or TNF-α (N≥11). HUVEC (1×106 cells/well) were incubated with CON or SCA PLTs (1×108 PLTs/well) for 4 h (37°C, 5% CO2), or with TNF-α, (10 ng/ml; 3 h, 37°C, 5% CO2). Expression of ICAM-1, E-selectin or VCAM-1 was analyzed on HUVEC (10 000 cells) by flow cytometry using anti-CD54-PE, CD62E-APC and CD106-FITC. **, p<0.01; ***, p<0.001, compared to basal (N≥16).
Figure 4.
Transwell inserts reverse the effects of SCA platelets on endothelial adhesion molecules expression.
Surface expressions of (A) ICAM-1 (CD54) and (B) E-selectin (CD62E) adhesion molecules on HUVEC (1×106 cells/well) following culture in the presence of healthy control (CON; N≥6; 1×108 PLTs/well) PLTs or steady-state sickle cell anemia (SCA; N ≥12; 1×108 PLTs/well) PLTs and in the presence and absence of transwell inserts (0.4 µm pores). Expressions of ICAM-1 and E-selectin were evaluated by flow cytometry using anti-CD54-PE and CD62E-APC. *, p<0.01; ***, p<0.001, compared to basal (N = 9).
Figure 5.
Release of IL-8 from co-cultures of HUVEC and PLTs.
Levels of IL-8 (ng/ml) were quantified in supernatants of co-cultures of HUVEC (1×106 cells/well) (4 h in suspension, 37°C, 5% CO2) in direct contact with CON (n = 27) or SCA PLTs (1×108 PLTs/well; n = 44), (4 h, 37°C, 5% CO2); or following pre-incubation of HUVEC with TNF-α (10 ng/ml; N = 24; 3 h, 37°C, 5% CO2). ***, P<0.001, compared to basal (N = 24); +++, P<0.001, compared to all groups.
Figure 6.
Effect of SCA platelets on endothelial gene expression.
Relative gene expression of (A) ICAM1, (B) NFKB1, (C) RELA, (D) CHUK and (E) IKKBETA in HUVEC after co-culture with PLTs. HUVEC (1×106 cells/well) were co-cultured in direct contact with CON (N = 10) or SCA PLTs (N≥24; 1×108 PLTs/well) (4 h, 37°C, 5% CO2); or pre-incubated with HUVEC with TNF-α (10 ng/ml; N≥11; 3 h, 37°C, 5% CO2). Relative gene expressions were determined by qPCR and expressed relative to ACTB and GAPDH expressions. *, P<0.05; **,P<0.01, compared to basal (N≥11). +++, P<0.001, compared to all groups.
Figure 7.
Effect of inhibition of the NF-κB pathway on the induction of endothelial adhesion molecule expression by SCA PLTs.
The expressions of (A) ICAM-1 (CD54) and (B) E-selectin (CD62E) were determined by flow cytometry on HUVEC (1×106 cells/well) following co-culture in direct contact with CON PLTs (N≥13) or SCA PLTs (1×108 PLTs/well; N≥14) in the presence or absence of BAY 11-7082 (20 µM; 4 h, 37°C, 5% CO2). *, P<0.05; **, P<0.01, compared to basal (N≥12).