Figure 4.

Dectin-1/Syk mediates the potentiating effects of Galectin-3 on platelet activation. (A) Galectin-3 enhanced phosphorylation of Src, Dectin-1, and PLCγ, as well as increased binding of p-Syk to Dectin-1 in human platelets. Washed platelets from healthy subjects were pre-treated with recombinant Galectin-3 (20 μg/L) for different times, as indicated. Representative results and summary data (see Supplementary material online, Figure S10A) of four experiments are presented. (B) Galectin-3 increased Ca²⁺ influx induced by different agonists. Washed platelets from healthy subjects loaded with Fura-2 were incubated with recombinant Galectin-3 (20 μg/L) for 5 min and measured for 300 s with collagen (0.2 μg/mL), thrombin (0.02 U/mL), or adenosine diphosphate (4.0 μM). Representative results and summary data (see Supplementary material online, Figure S10B) of four experiments are presented. (C) Galectin-3 increased p-PKC substrates induced by different agonists. Washed platelets from healthy subjects were pre-treated with recombinant Galectin-3 (20 μg/L) for 5 min and then stimulated with collagen (0.2 μg/mL), thrombin (0.02 U/mL), or adenosine diphosphate (4.0 μM) for 5 min. Red arrows mark the specific bands of p-PKC substrates. Representative results and summary data (see Supplementary material online, Figure S10C) of four experiments are presented. (D) Galectin-3 increased ROS production induced by different agonists. Washed platelets from healthy subjects loaded with DCFDA (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate) were incubated with recombinant Galectin-3 (20 μg/L) for 5 min and then stimulated with collagen (0.2 μg/mL), thrombin (0.02 U/mL), or adenosine diphosphate (4.0 μM) for 5 min. The DCF fluorescence intensity was measured, and the levels of ROS were standardized to the percentage observed in the resting platelets. Summary data of four experiments are presented. (E-F) Galectin-3-potentiated platelet aggregation and clot reraction were abolished by inhibitors targeting Dectin-1/Syk signalling downstream of Galectin-3. Washed platelets from healthy subjects were pre-treated with 10 μg/mL Dectin-1 Ab (Dectin-1 blocking antibody), 10 μM PP2 (Src inhibitor), 1 μM R406 (Syk inhibitor), 10 μM U73122 (PLCγ inhibitor), 50 μM 2-APB (Ca²⁺ inhibitor), 10 μM Ro 31-8220 (PKC inhibitor) or 10 μM VAS2870 (ROS inhibitor) for 10 min and then treated with 20 μg/L recombinant Galectin-3 or vehicle for 5 min. Platelet aggregation was initiated by 0.02 U/mL thrombin, and clot retraction was initiated by 1.0 U/mL thrombin. Representative results and summary data (see Supplementary material online, Figure S12) of four experiments are presented. Statistical analyses were performed using one-way ANOVA followed by Dunnett's multiple comparison test in (A) and Sidak's multiple comparison test in (F), and two-way ANOVA followed by Sidak's multiple comparisons test in (B), (C), and (D). Repeated measures one-way ANOVA followed by Sidak's multiple comparison test was performed in (E). NS, no significance; *P < 0.05; **P < 0.01.