FIGURE 6. Binding affinity and conformational preference of αIIbβ3 inhibitors.
(A) Binding affinity of FITC-echistatin for αIIbβ3 wild type (WT) and αIIbβ3_N305T transfectants determined using 5 nM Alexa647-MBC319.4 Fab in L15 medium with 0.1% BSA. Kd was obtained by fitting to Eq. 3 (Methods). Errors are standard fitting errors from the nonlinear least square fits. (B-C) Competition of representative αIIbβ3 inhibitors with FITC-echistatin (concentration on y axis) for binding to αIIbβ3 WT (B) and to αIIbβ3_N305T (C) on transfectants in L15 medium with 0.1% BSA. Binding assays shown here and in Fig. S3 with other inhibitors were determined multiple times with similar results. Inhibitor concentration range used in the experiment shown was designed based on previous competitive binding curves. (D) Binding affinities determined from competitive binding assays with nonlinear least square fits and error propagation (Eq.4, Methods). (E-F) Effect of αIIbβ3 inhibitors on integrin extension measured with the binding of 20 nM Alexa647-MBC319.4 Fab to αIIbβ3 WT (E) and αIIbβ3_N305T (F) transfectants by flow cytometry without washing in L15 medium with 0.1% BSA. Inhibitors were used at 100x their Kd values for αIIbβ3 WT reported in Panel D. Background MFI of 20 nM Alexa647-MBC319.4 Fab in presence of 2 μM unlabeled MBC319.4 antibody was subtracted from the MFI values of cells treated with or without inhibitors. Binding was measured three times; data show mean and standard deviation. Unpaired two-tailed student’s t-test was between the inhibitor and no drug groups: *: p<0.05; **: p<0.01; ***: p<0.001. See also Figures S3 and S4.