Binding of CLEC4M-Fc chimera to VWF. (A-F) Binding of CLEC4M to VWF was measured with a modified ELISA using immobilized VWF or CLEC4M-Fc. Receptors were blocked with mannan (1 mg/mL) for 30 minutes before incubation with VWF. (A) Binding of soluble VWF (10 µg/mL) to immobilized CLEC4M-Fc (10 µg/mL) and soluble CLEC4M-Fc and DC-SIGN-Fc (10 µg/mL) to immobilized Humate-P (10 µg/mL). (B) Dose-response curve of soluble CLEC4M-Fc and DC-SIGN-Fc (0.1-20 µg/mL) binding immobilized Humate-P (1 µg/mL). (C) Binding of CLEC4M and DC-SIGN to FVIII-free plasma-derived VWF, recombinant human VWF (D), and deglycosylated Humate-P (E) was assessed. (F) Calcium dependence, reversibility, and competition for soluble Humate-P were measured. N = 4 to 10 independent experiments; ± SE, *P < .05; **P < .001.