GPVI-ligation triggers aldose reductase activation. (A) Detection of aldose reductase (AR) in platelets by Western blot analysis. Cellular lysates from platelets treated with control IgG (RmC7H8) or GPVI antibody HGP4C9 (1 μg/mL) for 10 minutes were obtained, and AR expression (36-kDa protein) was determined by Western blot analysis. (B) Aldose reductase activity was measured as described previously by photometric quantification of NADPH reduction.²²  AR activity was normalized to protein content as measured by BCA assay and expressed in milliunits per microgram (nanomoles NADPH oxidized per minute per microgram of protein). For inhibition of aldose reductase (AR) platelets were preincubated with 10μM epalrestat for 10 minutes before stimulation with HGP4C9 (n = 6, mean ± SEM, *P < .05 for HGP4C9 vs IgG [solvent] and **P < .01 for HGP4C9 with solvent vs epalrestat). (C) Individual data of the human blood donors indicate the effects of platelet stimulation by HGP4C9 compared with control IgG. (1) Individual effects and (2) mean ± SEM (n = 6, *P < .05). (D) Activation of GPVI induces platelet aggregation involving aldose reductase. Aggregation of washed human platelets was induced by the GPVI antibody HGP4C9 (1 μg/mL), and light transmission was assessed in the presence of different aldose reductase inhibitors (epalrestat or quercetin) or solvent. Results are shown as aggregation in percentage of light transmission (n = 4-6, mean ± SEM, *P < .01 for solvent vs 10μM epalrestat and vs 10μM quercetin).