Lactadherin is present in circulating platelet-derived microvesicles in normal human plasma. (A) Microvesicles, isolated from normal human plasma by centrifugation, were incubated with PE-labeled anti-CD42b (2.5 μg/mL) and FITC-antilactadherin antibody L688 (5 μg/mL) or an FITC-labeled irrelevant control antibody. The CD42b-expressing particles were gated and analyzed for FITC fluorescence. (B) Immunoblot of microvesicle-associated lactadherin. Protein A + G agarose beads (100 μL) were incubated overnight at 4°C with 100 μg rabbit polyclonal anti-IIb/IIIa antibody or an irrelevant rabbit control antibody. The beads were washed and incubated with 5 mL platelet-poor plasma and washed. Bound proteins were eluted in 50 μL 1% SDS, subjected to SDS-PAGE, transferred to PVDF membrane, and probed with the monoclonal antibody to lactadherin L688²⁹  and developed by a peroxidase-labeled goat anti–mouse antibody (1/2000 dilution) and chloronaphthol (0.3 mg/mL) and H₂O₂ (0.03%). (Lane 1) Anti-IIb/IIIa anti-body and (lane 2) irrelevant control antibody. (Lane 1) Immunoprecipitation with anti-IIb/IIIa and (lane 2) irrelevant control antibody.