Effect of GSSG on platelet aggregation and effect of platelets on GSSG. (A) Gel-filtered platelets were incubated for 1 minute at 37° C with the indicated concentrations of GSSG. Collagen was then added at a concentration insufficient to induce aggregation by itself. (B) Collagen alone caused partial aggregation that a low concentration of GSSG (2 μM) further potentiated. These experiments were performed 3 times with similar results. (C-E) Gel-filtered platelets were incubated with GSSG, or under other conditions, and total sulfhydryl (SH) content in the sample was determined using DTNB as described.⁷  (C) Results of platelets incubated with GSSG. Platelets alone are in column 1. Column 2 shows the results of platelets preincubated with GSSG (5 μM) for 15 minutes at 24° C before the DTNB was added. Column 3 shows the results for GSH (10 μM) with platelets (Plat/GSH). For these experiments, n = 5. (D) The lack of effect of platelets on cystine (disulfide of cysteine) is seen. Column 1 again shows the amount of SH in platelets. Column 2 shows the SH content of platelets incubated with 5 μM cystine (Plat/cystine). Also shown are platelets with cysteine (10 μM; Plat/cysteine). For these experiments, n = 7. (E) The effect of NADPH oxidase inhibitors on conversion of GSSG to GSH is seen. The first column shows the SH content of platelets. Column 2 is the SH content of the platelet/GSSG mixture; columns 3 and 4 are the same as column 2 except that the flavoprotein NADPH oxidase inhibitors DPI (10 μM) or apocynin (100 μM; Apo) were added to the platelets for 30 minutes prior to the addition of GSSG. For these experiments, n = 2 to 4 for each sample. The results are ± 1 SE.