Quantification of platelet CLEC-2 expression. The number of surface copies of CLEC-2 per platelet was determined using the mouse α-human CLEC-2 antibody AYP1 and the Platelet Calibrator Kit from Biocytex. (A) Washed platelets were preincubated with 2.5 µg/mL AYP1 (saturating concentration) at room temperature for indicated times and subsequently incubated with a FITC-conjugated α-mouse antibody for another 15 minutes. Flow cytometric analysis shows that binding of AYP1 remains stable over the tested time period. (B) Calibrator beads, coated with batch-defined increasing concentrations of mouse IgG1 antibody molecules (i, 4400; ii, 29 000; iii, 10 800), were stained with FITC-conjugated α-mouse antibody and analyzed by flow cytometry. (C) The geometric mean fluorescence intensity (MFI) of the 3 bead populations were plotted against the corresponding number of mouse IgG1 molecules. Linear regression revealed an R2 of 0.996. (D) Washed platelets were preincubated with AYP1 for 30 minutes at room temperature and incubated with FITC-conjugated α-mouse antibody. The MFI was determined by flow cytometry and used to quantify surface expression of CLEC-2 by extrapolation from the linear regression line of C. CLEC-2 copy number was determined for ten donors of diverse ethnic backgrounds (23-55 years of age) with a mean of 2016 ± 239 (mean ± standard deviation).
