Platelet-derived CCL5 enhances proplatelet production. (A) Generation of activated platelet releasate. Platelet number was counted using a fluorescence-activated cell sorter and adjusted to 2 × 10⁸/mL. The resting state of platelets was confirmed by P-selectin antibody (BD Biosciences) labeling by flow cytometry. Platelets were either activated with 25 μM TRAP (Thrombin Receptor Activator Peptide, Sigma-Aldrich) or incubated with vehicle control for 10 minutes at 37°C. The resulting supernatant, or “releasate,” was separated from the cell pellet by centrifugation and used in subsequent experiments. (B-C) MKs from fetal liver cultures on day 4 of maturation were resuspended in 300 μL TRAP-activated or unactivated platelet releasate (with or without addition of anti-CCL5 antibody). 100 nM maraviroc (MIR) was added to indicated cultures 30 minutes prior to resuspension in platelet releasate. Proplatelet production from MKs was manually quantified after 6 hours based on images generated from a Nikon TE-2000-E Microscope (Nikon) equipped with a 20× (0.3 numerical aperture) Plan-Fluoro objective, using a Hamamatsu charged-coupled device camera, as previously described.^14-16  Briefly, for each replicate, at least 100 cells per condition were counted and scored as either “round” or “proplatelet-producing.” n = 3-6; *P < .05, **P < .01, with data plotted as mean and standard error of the mean and statistical analysis done by 1-way ANOVA with Tukey’s multiple comparisons test. Representative images of proplatelet formation are shown in panel C, indicating enhanced, long proplatelet strings with the addition of TRAP-activated platelet releasate. (D) Platelets were prepared as above and activated with 25 μM TRAP, 25 μM ADP (Biodata), 100 μM Thromboxane A₂ (Caymen), or 3 × 10⁶/mL MCF-7 breast tumor cells (ATCC). CCL5 in releasate was measured using the Quantikine human CCL5 enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (R&D Systems). n = 3; *P < .05, **P < .01, with data plotted as mean and standard error of the mean and statistical analysis done by 1-way ANOVA showing differences compared with resting platelet control. (E) Platelets and mouse MKs were isolated and prepared as previously described. Human MKs were isolated from umbilical cord blood collected with institutional review board approval from healthy full-term neonates (38-42 weeks gestation) at Brigham and Women’s Hospital Labor and Delivery. Briefly, CD34⁺ cells were then isolated using a positive magnetic selection system (Miltenyi Biotec) and plated in 24-well plates at 1 × 10⁵ cells/mL and cultured in serum-free medium with rTPO (50 ng/mL, PeproTech), with twice-weekly medium changes for 14 days. Live-cell number was quantified twice weekly by staining with 0.4% Trypan blue. For immunofluorescence, samples were fixed in 4% formaldehyde and centrifuged onto poly-l-lysine (1 μg/mL)-coated coverslips, permeabilized with 0.5% Triton-X-100, and blocked in blocking buffer.¹⁸  Samples were examined with a Zeiss Axiovert 200 (Carl Zeiss, Thornwood, NY) equipped with a 63× or 100× (1.4 numerical aperture) Plan-ApoChromat oil-immersion objective, and images were obtained and analyzed using Metamorph software (Molecular Devices, Sunnyvale, CA) and ImageJ (National Institutes of Health; http://rsb.info.nih.gov/ij/). Scale bars represent 2 μm (platelets) and 20 μm (MKs); green indicates CCL5. In platelet samples only, red indicates β-tubulin. In MK samples only, blue indicates Hoechst (nucleus). (F-G) Surface expression of CCR5 was determined on mouse and human MKs and platelets using a phycoerythrin-conjugated anti-human/mouse CCR5 antibody (R&D Systems) compared with an isotype control. (G) Representative histograms are shown with CCR5-positive staining (red) overlayed onto isotype control (gray). (H) CCR5 expression was quantified. n = 3. Ab, antibody; plt, platelet; rlsate, releasate.