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).