Quantification of VWF internalization by CLEC4M-expressing cells. (A-E) To quantify binding and internalization of VWF by cells stably expressing CLEC4M, cells were incubated with Humate-P, washed, and lysed. Levels of VWF in the cell lysate were quantified by ELISA. (A) Flow cytometric confirmation of CLEC4M expression by stable cell lines. (B-C) Levels of VWF in the cell lysate were measured as a dose response and as a time course. (D) Preincubation of CLEC4M-expressing cells with mannan (100 µg/mL) partially attenuated VWF binding and internalization. (E) Internalization of VWF by CLEC4M was confirmed by incubating CLEC4M-expressing cells with 2 U of VWF for 24 hours, and measuring levels of VWF in cell lysates prepared from trypsinized and nontrypsinized cells. N = 4 to 6 independent experiments; ± SE, *P < .05; **P < .001.