Specificity of [¹²⁵I]CyPB binding to platelets. (A) Competitive binding assays with cyclophilin isoforms and synthetic peptides copying specific sequences of CyPB. Platelets were incubated in the presence of 50 nmol/L [¹²⁵I]CyPB and increasing concentrations of unlabeled CyPA (▪), CyPB (•), CyPC (⧫), N-terminal peptide of CyPB (○), C-terminal peptide of CyPB (□), or RGDS peptide (⋆). After washing, the amounts of remaining surface-bound [¹²⁵I]CyPB, expressed as a percentage of the ligand bound in the absence of competitors, are plotted against the molar ratios of [¹²⁵I]CyPB to the competitors. Data are expressed as mean values from 3 separate experiments conducted with platelets from different donors. (B) Sensitivity of CyPB binding to cyclosporine derivatives, protamine, and GAG-degrading enzymes. Platelets were incubated with of 50 nmol/L [¹²⁵I]CyPB in the absence (1) or presence of CsA 500 nmol/L (2); CsA 5 μmol/L (3); CsG 500 nmol/L (4); CsG 5 μmol/L (5), CsH 500 nmol/L (6); CsH 5 μmol/L (7); protamine 500 nmol/L (8); or protamine 5 μmol/L (9). In the last cases, platelets were first pretreated with heparinase type I (10), chondroitinase ABC (11), or both (12), and directly used for binding experiments. After washing, the amounts of remaining surface-bound [¹²⁵I]CyPB were expressed as a percentage of the ligand bound in the absence of any treatment. Data are mean values ± SEM from 3 separate experiments conducted with platelets from different donors.