Figure 3.
Galectin-3 expressed on tumor cells supports GPVI-dependent platelet adhesion. (A) Prediction of binding affinity between galectin-3 (red) and GPVI (blue). Graphs correspond to ΔGbind = −5.91 kcal/mol and KD = 44.89 μM, respectively. Potential of mean force (PMF) values were extracted by the weighted histogram analysis method, which yields the binding free energy (ΔGbind) for the binding and unbinding processes. The ΔGbind value was calculated as the difference between the highest and lowest values of the PMF curve. The KD of the protein–protein complex was calculated from the ΔGbind value according to the following equation: KD = exp(ΔGbind/R · T), where R (gas constant) is 1.98 cal(mol·K)−1 and T (room temperature) is 298.15 Kelvins (K). The Boltzmann (sigmoidal) nonlinear fitting [R2 = 0.85; RMSE (root mean square error) = 0.67; RSS (residual sum of squares) = 88.31] of the data set is shown to assess the PMF elevation pattern with the increase of reaction coordinate (ξ), using the scaled Levenberg-Marquardt algorithm with tolerance = 0.0001. (B-C) Knockout of the galectin-3 gene (Lgals3 KO) expression in MC38 and AT-3 cells. (B) Galectin-3 mRNA expression was determined by quantitative reverse transcription polymerase chain reaction and normalized with a messenger RNA level of glyceraldehyde-3-phosphate dehydrogenase. Data are presented as the mean ± standard deviation (SD) of 4 separate cell culture experiments; *P < .05, by Mann-Whitney test. (C) Representative immunofluorescence images of galectin-3 (Lgals3 KO) knockout MC38 and AT-3 cells or cells expressing galectin-3 gene (Ctrl: control), with an anti-galectin-3 antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The bar represents 20 µm. (D-E) Quantification of the fluorescence signal corresponding to the amount of WT (D-E) and Gp6−/− (D) mouse platelets adhering to (D-E) MC38 and AT-3 Ctrl and Lgals3 KO cancer cells. (D) Data are presented as the mean ± SD; n = 3 mice per group. MC38 Ctrl/WT platelets (plts; 3.607 ± 0.6198); MC38 Ctrl/Gp6−/− plts (1.593 ± 0.2774); MC38 Lgals3 KO/WT plts (1.6 ± 0.2402); and MC38 Lgals3 KO/Gp6−/− plts (1.423 ± 0.1665). AT-3 Ctrl/WT plts (5.37 ± 0.3005); AT-3 Ctrl/Gp6−/− plts (1.897 ± 0.4944); AT-3 Lgals3 KO/WT plts (1.817 ± 0.586); and AT-3 Lgals3 KO/Gp6−/− plts (1.683 ± 0.486). ***P < .001; ****P < .0001, 1-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (E) Similar experiments were performed with WT platelets in the absence or presence of JAQ1 F(abʹ)2 or an irrelevant control F(abʹ)2 antibody. Data are presented as the mean ± SD; n = 3 mice per group. MC38 Ctrl/Ctrl F(abʹ)2 plts (5.017 ± 0.2055); MC38 Ctrl/JAQ1 F(abʹ)2 plts (2.437 ± 0.493); MC38 Lgals3 KO/Ctrl F(abʹ)2 plts (2.757 ± 0.2139); and MC38 Lgals3 KO/JAQ1 F(abʹ)2 plts (2.273 ± 0.4366). AT-3 Ctrl/Ctrl F(abʹ)2 plts (5.313 ± 0.5326); AT-3 Ctrl/JAQ1 F(abʹ)2 plts (2.38 ± 1.057); AT-3 Lgals3 KO/Ctrl F(abʹ)2 plts (1.967 ± 0.2259); and AT-3 Lgals3 KO/JAQ1 F(abʹ)2 plts (2.01 ± 0.1825). **P < .01; ***P < .001; ****P < .0001, by 1-way ANOVA, followed by Tukey’s post hoc test. (F,H) Representative immunofluorescence microscopy images of mouse (WT and Gp6−/−) (F) and human (H; top; Ctrl, healthy subject; hGP6+/−, human patient with heterozygous mutation; and hGP6−/−, human patient with homozygous mutation) platelets adhering to recombinant galectin-3 under static conditions. The bar represents 20 µm. (H, bottom). Representative immunofluorescence images of GPVI-Fc–conjugated fluorescent microspheres adhering on galectin-1–, galectin-3– and collagen I–coated surfaces. (G,I) Quantification of adherent mouse (G) and human (I) platelets under static conditions on galectin-3. (G) Mean ± SD; n = 4 mice per group; *P < .05, by Mann-Whitney test. (I) Mean ± SD. Ctrl, n = 5; hGP6+/−, n = 2; and hGP6−/−, n = 3. (J) Binding of dimeric GPVI to galectin-3. GPVI–Fc-Alexa-488 was added to microwells coated with galectin-1, galectin-3, collagen I, or bovine serum albumin (BSA). Protein binding was assessed by measuring the fluorescence intensity (FI) at 488 nm. Data are presented as the mean ± SD of 4 separate experiments. *P < .05; **P < .01, by 1-way ANOVA, followed by Tukey’s post hoc test.

Galectin-3 expressed on tumor cells supports GPVI-dependent platelet adhesion. (A) Prediction of binding affinity between galectin-3 (red) and GPVI (blue). Graphs correspond to ΔGbind = −5.91 kcal/mol and KD = 44.89 μM, respectively. Potential of mean force (PMF) values were extracted by the weighted histogram analysis method, which yields the binding free energy (ΔGbind) for the binding and unbinding processes. The ΔGbind value was calculated as the difference between the highest and lowest values of the PMF curve. The KD of the protein–protein complex was calculated from the ΔGbind value according to the following equation: KD = exp(ΔGbind/R · T), where R (gas constant) is 1.98 cal(mol·K)−1 and T (room temperature) is 298.15 Kelvins (K). The Boltzmann (sigmoidal) nonlinear fitting [R2 = 0.85; RMSE (root mean square error) = 0.67; RSS (residual sum of squares) = 88.31] of the data set is shown to assess the PMF elevation pattern with the increase of reaction coordinate (ξ), using the scaled Levenberg-Marquardt algorithm with tolerance = 0.0001. (B-C) Knockout of the galectin-3 gene (Lgals3 KO) expression in MC38 and AT-3 cells. (B) Galectin-3 mRNA expression was determined by quantitative reverse transcription polymerase chain reaction and normalized with a messenger RNA level of glyceraldehyde-3-phosphate dehydrogenase. Data are presented as the mean ± standard deviation (SD) of 4 separate cell culture experiments; *P < .05, by Mann-Whitney test. (C) Representative immunofluorescence images of galectin-3 (Lgals3 KO) knockout MC38 and AT-3 cells or cells expressing galectin-3 gene (Ctrl: control), with an anti-galectin-3 antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The bar represents 20 µm. (D-E) Quantification of the fluorescence signal corresponding to the amount of WT (D-E) and Gp6−/− (D) mouse platelets adhering to (D-E) MC38 and AT-3 Ctrl and Lgals3 KO cancer cells. (D) Data are presented as the mean ± SD; n = 3 mice per group. MC38 Ctrl/WT platelets (plts; 3.607 ± 0.6198); MC38 Ctrl/Gp6−/− plts (1.593 ± 0.2774); MC38 Lgals3 KO/WT plts (1.6 ± 0.2402); and MC38 Lgals3 KO/Gp6−/− plts (1.423 ± 0.1665). AT-3 Ctrl/WT plts (5.37 ± 0.3005); AT-3 Ctrl/Gp6−/− plts (1.897 ± 0.4944); AT-3 Lgals3 KO/WT plts (1.817 ± 0.586); and AT-3 Lgals3 KO/Gp6−/− plts (1.683 ± 0.486). ***P < .001; ****P < .0001, 1-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (E) Similar experiments were performed with WT platelets in the absence or presence of JAQ1 F(abʹ)2 or an irrelevant control F(abʹ)2 antibody. Data are presented as the mean ± SD; n = 3 mice per group. MC38 Ctrl/Ctrl F(abʹ)2 plts (5.017 ± 0.2055); MC38 Ctrl/JAQ1 F(abʹ)2 plts (2.437 ± 0.493); MC38 Lgals3 KO/Ctrl F(abʹ)2 plts (2.757 ± 0.2139); and MC38 Lgals3 KO/JAQ1 F(abʹ)2 plts (2.273 ± 0.4366). AT-3 Ctrl/Ctrl F(abʹ)2 plts (5.313 ± 0.5326); AT-3 Ctrl/JAQ1 F(abʹ)2 plts (2.38 ± 1.057); AT-3 Lgals3 KO/Ctrl F(abʹ)2 plts (1.967 ± 0.2259); and AT-3 Lgals3 KO/JAQ1 F(abʹ)2 plts (2.01 ± 0.1825). **P < .01; ***P < .001; ****P < .0001, by 1-way ANOVA, followed by Tukey’s post hoc test. (F,H) Representative immunofluorescence microscopy images of mouse (WT and Gp6−/−) (F) and human (H; top; Ctrl, healthy subject; hGP6+/−, human patient with heterozygous mutation; and hGP6−/−, human patient with homozygous mutation) platelets adhering to recombinant galectin-3 under static conditions. The bar represents 20 µm. (H, bottom). Representative immunofluorescence images of GPVI-Fc–conjugated fluorescent microspheres adhering on galectin-1–, galectin-3– and collagen I–coated surfaces. (G,I) Quantification of adherent mouse (G) and human (I) platelets under static conditions on galectin-3. (G) Mean ± SD; n = 4 mice per group; *P < .05, by Mann-Whitney test. (I) Mean ± SD. Ctrl, n = 5; hGP6+/−, n = 2; and hGP6−/−, n = 3. (J) Binding of dimeric GPVI to galectin-3. GPVI–Fc-Alexa-488 was added to microwells coated with galectin-1, galectin-3, collagen I, or bovine serum albumin (BSA). Protein binding was assessed by measuring the fluorescence intensity (FI) at 488 nm. Data are presented as the mean ± SD of 4 separate experiments. *P < .05; **P < .01, by 1-way ANOVA, followed by Tukey’s post hoc test.

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