Figure 1.
Identification of platelets and leukocytes positive for dual cell-specific antigens.
A. Representative scatter plot of the gate used to identify platelets by size. B. Quadrants derived from platelet gate of blood stained with CD41-PE plus IGg-FITC (matched isotype control). C. Quadrants derived from platelet gate of blood stained with CD41-PE plus CD45-FITC (leukocyte-specific antibody). D & E. Cumulative data of platelets expressing leukocyte antigen (D) and leukocytes expressing platelet antigen (E) after incubation of blood with LPS (500 ng/mL) for up to 60 minutes. Data are derived from positive quadrants of platelet and leukocytes gates of scatter plots. WT mice (open bars); dTLR4 mice (closed bars). Data are expressed as mean ± SEM (n = 8/group) of fold increases from the vehicle-treated group at the same time point. *P<0.05 vs. saline-treated group at the same time point.
Figure 2.
Representative dark-field micrographs of platelets from a WT mouse taken immediately before or after 30 and 60 min.
incubation of the blood with 500 ng/ml LPS. Prior to LPS application, platelet aggregates (of more than 5 platelets) were absent, and most platelets were discoid shaped (Left panels). At 30 and 60 minutes incubation with saline, platelet morphology did not change (Top panels); whereas, large aggregates of irregularly-shaped platelets were observed in those incubated with LPS (bottom panels, inset shows platelets with pseudopodia). Each bar represents 5 µm. Similar results were observed with blood from two additional animals (n = 3).
Table 1.
Expression of PS aggregates and plasma MV before and after treatment with LPS (500 ng/ml for 60 minutes).
Figure 3.
Quantification of platelets positive for leukocyte antigens using FASCalibur™ flow cytometry set to the platelet gate after incubation with inhibitory peptides.
Cumulative changes in number of platelets positive for leukocyte antigens after incubation of whole blood with saline or LPS (500 ng/mL; for 60 min), in the absence or presence of control (100 µM) or inhibitory peptides MyD88 and TRIF (100 µM each). Data are expressed as mean ± SEM (n = 8/group). *P<0.05 vs. saline-treated group, ‡P<0.01 vs. control peptide-treated group incubated with LPS.
Figure 4.
Sub-classes of leukocytes and apoptotic bodies expressing platelet antigen derived from blood of male mice seven days after a single intravenous injection of LPS (25 ng/gm body weight) or vehicle.
A. Scatter plot of leukocyte triggered by CD45-APC. B. Quadrants derived from granulocyte gate. C. Quadrants derived from monocyte gate. D. Quadrants derived from lymphocyte gate. E. Quadrants derived from apoptotic bodies. F. Cumulative data expressed as mean ± SEM (n = 5) of fold increase from the vehicle-treated group of each sub-class of leukocytes positive for platelet antigen. Asterisks denote statistical significance (p<0.05) from baseline values, i.e. prior to the intravenous injections.