The extracellular domain of CLEC-2 is required for GPIbα-mediated platelet activation. (A) Schematic for the construction of a chimeric CLEC-2 that fused human IgG Fc with the CLEC-2 extracellular domain (CLEC-2/FC). hFC, human IgG Fc control; N, N-terminus; C, C-terminus. (B) Recombinant CLEC-2 inhibited VWF-induced platelet aggregation. Washed WT platelets were pretreated with Fc or CLEC-2/Fc at 37°C for 1 hour, and aggregation of platelets in response of human plasma VWF was measured. (C) Quantification of platelet aggregation shown in (B). (D) Activation of integrin αIIbβ3 on CLEC-2/Fc-treated platelets. Washed WT platelets were pretreated with Fc or CLEC-2/Fc at room temperature for 1 hour and then treated with VWF in the presence of botrocetin for 5 minutes at 37°C. Then, integrin αIIbβ3 activation was measured with antibody Jon/A by flow cytometry. (E) Recombinant CLEC-2 inhibited platelet aggregation on immobilized VWF under flow. WT platelets were preincubated with Fc or CLEC-2/Fc (30 μg/mL) for 1 hour at 37°C and were then perfused over plates coated with VWF from human plasma (100 μg/mL) at 10 dyne/cm² for 10 minutes. Adherent platelets were fixed with 2% PFA and stained with anti-mouse CD41, followed by Alexa 488-conjugated secondary antibodies. Representative images were presented. (F) Quantification of aggregated platelets shown in (E). The data are representative of 3 independent experiments and represent mean ± SD. *P < .05.