Characterization of Co-HP. (A) ELISA-based IC₅₀ curves for the association between hPod-hFc2 and immobilized hCLEC-2–rFc2 in the presence of Co-HP. (B) (i) Interaction between CLEC-2 (green) and Co-HP (brown). Amino acid residues involved in the Co-HP–CLEC-2 interaction are labeled and highlighted in pink. (ii) Interaction between CLEC-2 (green) and Co-HP (brown). Four arginine residues used for the podoplanin or rhodocytin–CLEC-2 interactions reported to date are labeled and highlighted in red. Amino acid residues involved in the Co-HP–CLEC-2 interactions are highlighted in blue. (C) Flow cytometric analysis using wild-type (WT) and mutant hCLEC-2–hFc2 in binding sites for podoplanin, rhodocytin, or Co-HP (R107A, R118A, R152A, R157A, N120A, N210A, and K211A) and podoplanin-expressing CHO cells. R107A, R118A, R152A, R157A, N120A, N210A, and K211A indicate that arginine, asparagine, and lysine residues in the indicated positions of the recombinant CLEC-2 are substituted with alanine residues. Fill, Purified hFc2; line, purified wild-type or mutant hCLEC-2–hFc2. (D) Binding of wild-type or mutant hCLEC-2–rFc2 to podoplanin-expressing CHO cells. Results are expressed as the relative MFI ± SD (n = 4, panel C) compared with MFI of control rFc2 binding.