PDMV delivery of CD154 signal. (A) CD154^−/− mice were immunized on day (−1) and then injected on days 0 and 6 with activated platelet supernatant (AP Sup), PDMV pellet, or PDMV-poor supernatant from 5 × 10⁸ wild-type (B6) or CD154^−/− platelets, or 500 μg 1C10 intraperitoneally. Serum was collected on day 9 for quantification of total adenovirus-specific IgG using a commercially available mouse anti-adenovirus IgG standard. (B) PDMVs derived from 5 × 10⁸ B6 platelets were injected intravenously into CD154^−/− mice 24 hours after immunization with 10⁸ particles Ad-OVA; 10 μg/mL CD154 blocking antibody MR-1 was added to AP Sup before isolation of PDMVs and to PDMVs after resuspension. Mice were injected with 100 μg MR-1 just before receiving PDMV + MR-1. Serum was collected on day 7 for quantification of total adenovirus specific IgG production by ELISA. (C) PDMVs derived from 5 × 10⁸ platelets injected 24 hours after immunization. Serum collected on day 7. (D) Primary B cells were isolated from spleens of 8-week-old C57BL/6 mice by Percoll gradient enrichment and negative selection over magnetic beads (Miltenyi Biotec). 6 × 10⁵ B cells were plated in each well of a 96-well plate and PDMVs from 4.5 × 10⁸ wt or ko platelets added to each designated well, with or without anti-IgM, 5 wells per experimental condition. PDMVs and B cells were coincubated for 48 hours, with the final 6 hours in the presence of 1 μCi of ³H-T. Cells were harvested and thymidine incorporation measured (Wilcoxon rank sum test: *2-tailed P = .012, **2-tailed P = .019).