![Figure 1. The importance of the C-terminal 14-3-3-binding site of GPIbα in GPIb-mediated cellular response to thrombin. (A) CHO cells expressing endogenous PAR1 and wild-type human GPIb-IX (1b9), GPIb-IX with a 5-amino acid truncation in the C-terminal 14-3-3-binding site of GPIbα (Δ605), GPIb-IX with a triple tyrosine to phenylalanine mutation (Y276 278,279 F, FFF) in the thrombin binding site of GPIbα, and control CHO cells expressing PAR1 but not GPIb-IX were each loaded with a calcium-sensitive fluorescent dye, FLIPR Calcium 5 reagent, and then stimulated with 3 or 6 nM thrombin. Kinetic changes from baseline calcium fluorescence were recorded and plotted as a fluorescence ratio (FR).4 (B) Quantification of the area under the curves (AUC) shown in A from 4 independent experiments (mean ± standard error of the mean [SEM]; *P < .05 and **P < .01, 1-way analysis of variance [ANOVA]). (C-E) Flow cytometric analysis of the binding of (C) an anti-PAR1 monoclonal antibody WEDE15, (D) an anti-GPIb-IX complex monoclonal antibody SZ1, or (E) an anti-GPIbα monoclonal antibody SZ2 as detected using Alexa Fluor 488–labeled goat anti-mouse IgG to control CHO cells, 1b9, Δ605, and FFF cells.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/127/5/10.1182_blood-2015-04-638387/4/626f1.jpeg?Expires=1735813451&Signature=Y5ZqT4qwpfkjVs~sc7CBt524vHTcb3wJ0BmOfhnhY6Mc2fNTzExZcJLSXMdKw6omnykEQ7KUsvBukLFVLQZDPaiYe2Jz-oveXYhwXBm2t36dwz-keFwOQetQ1MKAO1cA5Qa3YJUT9vcDjstQGCWMKZszVU-XXA1Re0ZnKqnDlhN~muvCbFq8G0MLkWwuIXKtbdbbNNORerfFwS1KZtRKjt-dMQQQ1sXhkpLK7MlfdTw8~~7yS47W-hQEHuPFJklY-W7i0yubfTZfuolTbEZNeUfn2X72W0juOY3ZKyjeib8zT4OKxTin2MHQry-dTxJ~vIeaH0jrHwix5uCof6X08A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The importance of the C-terminal 14-3-3-binding site of GPIbα in GPIb-mediated cellular response to thrombin. (A) CHO cells expressing endogenous PAR1 and wild-type human GPIb-IX (1b9), GPIb-IX with a 5-amino acid truncation in the C-terminal 14-3-3-binding site of GPIbα (Δ605), GPIb-IX with a triple tyrosine to phenylalanine mutation (Y276 278,279 F, FFF) in the thrombin binding site of GPIbα, and control CHO cells expressing PAR1 but not GPIb-IX were each loaded with a calcium-sensitive fluorescent dye, FLIPR Calcium 5 reagent, and then stimulated with 3 or 6 nM thrombin. Kinetic changes from baseline calcium fluorescence were recorded and plotted as a fluorescence ratio (FR).4 (B) Quantification of the area under the curves (AUC) shown in A from 4 independent experiments (mean ± standard error of the mean [SEM]; *P < .05 and **P < .01, 1-way analysis of variance [ANOVA]). (C-E) Flow cytometric analysis of the binding of (C) an anti-PAR1 monoclonal antibody WEDE15, (D) an anti-GPIb-IX complex monoclonal antibody SZ1, or (E) an anti-GPIbα monoclonal antibody SZ2 as detected using Alexa Fluor 488–labeled goat anti-mouse IgG to control CHO cells, 1b9, Δ605, and FFF cells.
