Microparticles from activated platelets maintain CLEC-2, but lose GPVI expression. (A) Microparticles were isolated from fresh PRP as described in the methods section and (left) CD41⁺ microparticles were analyzed for (center) CLEC-2 and (right) GPVI expression using Alexa Fluor 488-conjugated α-CLEC-2 antibody AYP1 and α-GPVI antibody 1G5, respectively. True positivity was determined using isotype-matched controls. (B-C) CD41⁺ microparticles isolated from PRP from a 12-day megakaryocyte (MK) culture or from washed platelets (WP) that were incubated for 1 hour at 37°C with CRP (10 µg/mL) or AYP1 Fab (2.5 µg/mL) cross-linked with 20 µg/mL α-mouse Fab-specific F(ab)₂ fragments (xAYP1) were analyzed for (B) CLEC-2 and (C) GPVI expression. Data are presented as the ratio of MFI of CLEC-2 or GPVI over isotype controls (n = 4). (D-E) Analysis of microparticles from healthy controls and patients with rheumatoid arthritis. CD41⁺ microparticles isolated from fresh PRP were analyzed for (D) CLEC-2 and (E) GPVI expression. Data are presented as the ratio of MFI over isotype controls (mean ± standard deviation; n = 10).