Characterization of PDMVs. PDMVs were isolated and visualized by TEM and analyzed for total protein. (A) TEM images of whole platelets (top) and PDMV (bottom). Bar represents 100 nm. (B) Total protein analysis was performed on 5 × 108 unactivated platelets (UAP) and activated platelets (AP); and activated platelet supernatant (AP Sup), PDMV pellet (PDMV), and PDMV-poor supernatant (PDMV-poor Sup) made from 5 × 108 platelets. ***Total protein values (mg/mL) above each bar. (C) 105 MS-1 cells were plated in each well of a 24-well plate and allowed to grow to near confluence over 2 days. PDMVs from 4.5 × 108 CD154 wt or ko platelets, or 108 platelets were added to each well; 10 μg/mL of anti-CD154 and/or anti-TNF-α antibodies added to designated wells. The experiment was performed in triplicate. (D) Quantitative real-time PCR was performed using purchased primers for MCP-1 and 18S mRNA. Standard curve ranged from 10 μg/mL to 4.9 ng/mL.
