Fig 1.
Pro- and anti-apoptotic adaptor proteins of the extrinsic pathway of apoptosis are expressed in human platelets.
(A) Representative images of platelets co-stained for CD41 (red) with either DJ-1, FADD, TRAF2, TRADD, c-FLIP or DEDAF (all displayed in green). CD41 stains for integrin αIIb (GPIIb), a transmembrane glycoprotein expressed on the platelet surface and on megakaryocytes. Shown is one confocal plane, objective: 63x glycerol with a numerical aperture of 1.3. Images were processed by using confocal laser scanning microscope SP8 (Leica). Scale bar 20μm. (B) Identification of DJ-1, FADD, TRAF5, TRADD, c-FLIP, and DEDAF by flow cytometry. Results are presented as CD42a positive events. Importantly, TRAF2 (green) was only identified by confocal microscopy (A) and TRAF5 (blue) was only detected by flow cytometry analysis (B). We used IgG isotype as control.
Fig 2.
FADD and DJ-1 co-localize and possibly interact in human platelets.
(A) Representative confocal images show the co-localization of FADD (green) and DJ-1 (red) resulting in a yellow stain (white arrows). Shown is one confocal plane, objective 63x glycerol with a numerical aperture of 1.3. Images were processed by using confocal laser scanning microscope SP8 (Leica). We also calculated a colocalization correlation coefficient Pearson’s R value of 0.88 with Image J software. Scale bar 10μm. (B) co-IP of FADD and DJ-1 in human PCs. As control the whole platelet lysate was immunoblotted (Input) and IgG controls were used to determine unspecific binding of the protein to the antibody (n = 3). Immunoblot 1 (WB 1 top): FADD probed; Immunoblot 2 (WB 2 bottom): DJ-1 probed.
Fig 3.
ABT-737 exposure disrupts the co-localization of DJ-1 with FADD and activates caspase-8 in platelets.
(A) Immunofluorescence co-staining of unstimulated and ABT-737 stimulated platelets for FADD (green) and DJ-1 (red). Merged images show that the moderate co-localization of DJ-1 and FADD (yellow, upper image) is disrupted in ABT-737 stimulated platelets (lower image). (B) Immunofluorescence co-staining of unstimulated and ABT-737 stimulated platelets for FADD (green) and caspase-8 (red). Merged images show increased co-localized expression of FADD and caspase-8 in ABT-737 stimulated platelets. Results are shown as one confocal plane, objective 63x glycerol with a numerical aperture of 1.3. Images were processed by using confocal laser scanning microscope SP8 (Leica). Scale bar 20μm. (C) Caspase-8 (procaspase-8 p53/54, first cleavage fragments p41/43, and the active p18 form), DJ-1 and Bcl-XL expression was assessed by Western blotting in ABT-737 treated platelets (lane 1) and in unstimulated cells (lane 2). Platelets were treated for 2h at 37°C. A representative immunoblotting image was selected (n = 3).
Fig 4.
TNF, FasL, and TWEAK treatment of human platelets does not show caspase- 3/7 activation and PS exposure.
Flow cytometry of washed platelets treated with TNF, FasL, TWEAK or ABT-737. (A) Platelets were labeled with CD42a-PE (Y-axis) and caspase- 3/7 positivity is shown as fluorescence intensities of FLICA positive events, measured as green fluorescent signal (X-axis). Percentages show double CD42a-PE and caspase-3/7 positive events. CD42a-PE positive only events (platelets) are shown on the left side of the graph. (B) PS exposure is shown as fluorescence intensities of AnnexinV (APC) positive platelets (Y-axis). Percentages show double CD42a-PE and AnnexinV positive events. CD42a-PE positive only events (platelets) are shown on the left side of the graph.
Fig 5.
ITP plasma treatment of platelets derived from healthy donors show no significant caspase- 3/7, and -8 activation.
Flow cytometry analyses of healthy platelets treated with ITP (n = 3), healthy (n = 2) and autologous plasma (n = 2) and A23187 (n = 2). Healthy platelets were incubated for 2h with ITP plasma at 37°C. FLICA% platelets are: (A) FLICA reagent, FAM-DEVD-FMK, irreversibly bound to active caspase- 3/7, and (B) FLICA reagent, FAM-LETD-FMK, irreversibly bound to active caspase- 8 (B), both measured as green fluorescent signal. Autologous plasma was used as a negative control. Statistical analyses were performed using one-way ANOVA followed by multiple comparisons tests to compare the mean ranks between groups. Data are presented as Standard Error of the Mean (SEM).
Fig 6.
Schematic overview of pro- and anti-apoptotic proteins of the extrinsic apoptosis pathway identified in human platelets (right) in comparison with nucleated cells (left).
In nucleated cells, several death receptors are known, e.g. TNFRI/II, Fas/CD95, DR 1–5, which could not be yet identified in human platelets. Grey box: adaptor-proteins of the extrinsic apoptosis pathway identified by our group and others, e.g. TRADD, TRAF2/5, cIAP1/2, FADD, DJ-1, NEMO, IKK1/2, NFκB, and NFκB1.