![Figure 2. Time-course studies on GSSG and platelets and the effect of GSSG on sulfhydryl labeling of αIIbβ3. (A) Time course of GSSG conversion to GSH at 37° C. In these studies GSSG was added to platelets for the indicated times, and then DTNB was added. Total SH in the sample was measured as described for Figure 1C. Each point in the graph represents the mean of duplicate samples. (B) GSSG (5 μM) was added either simultaneously (0 seconds) with a subthreshold concentration of collagen (0.1 μg/mL) or 1 minute (60 seconds) before the subthreshold dose of collagen. (Controls not shown demonstrated the stimulatory effect of GSH (10 μM) or GSH (10 μM) with GSSG [2 μM], added simultaneously with the same dose of collagen.) These experiments were performed a minimum of 2 times with similar results. (C) Platelets were incubated without GSH (lane 1), with GSH (10 μM; lane 2), or with GSSG (5 μM; lane 3) for 5 minutes at 24° C under nonstirring conditions. The sulfhydryl reagent 3-N-maleimidylpropionyl biotin (MPB; 50 μM) was added in excess of the GSH, and the labeling performed under conditions that cause disruption of the αIIbβ3 receptor (5 mM EDTA [ethylenediaminetetraacetic acid], 60 minutes, 37° C). Equal amounts of protein were added to lanes 1, 2, and 3. (D) Densitometry of the individual bands was used to obtain quantitative results. The first 2 columns show the ratio of labeling of the β3 subunit with GSH or GSSG to the β3 subunit without GSH (± 1 SE, n = 8). As a control for possible translocation of αIIbβ3 the third and fourth columns give the ratio of labeling using a label for primary amines, sulfosuccinimido-biotin (SSB) in the β3 subunit with GSH or GSSG to the β3 subunit without GSH (± 1 SE).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/104/5/10.1182_blood-2004-03-1097/5/zh80170465770002.jpeg?Expires=1735839698&Signature=SDclOP5KbFWIDsnX5~i7g1jWPOPS4M7EdaLsdiWNFPjezFwrCCqvOvTBkJQ7eZK56lNk7rBfrhNSBloGrNNbx1jssdNcYl4oZkO~D3o-Ypvm6~SZwU3B1J92RyV4aNxlG~wPhvwHrbFwI4MYBbnPlfPpRv~kMfyp26vi5PlvOy3Q9O5hxt0~ezoAxGwhARejivGYV1FsRBwV0H9tUEm~Jkqmouq9~Rfg1ggnWouMHFaLB4jjPMyuz7eSfM7L-A8gu1gNDubnMng25sMB2ov7lhWRf9uhxMLd9fa8F2CmBoDS1GzK3KC8U6hfw7GPSiwx9IbFPK67zrpXv9JlhLyk2g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Time-course studies on GSSG and platelets and the effect of GSSG on sulfhydryl labeling of αIIbβ3. (A) Time course of GSSG conversion to GSH at 37° C. In these studies GSSG was added to platelets for the indicated times, and then DTNB was added. Total SH in the sample was measured as described for Figure 1C. Each point in the graph represents the mean of duplicate samples. (B) GSSG (5 μM) was added either simultaneously (0 seconds) with a subthreshold concentration of collagen (0.1 μg/mL) or 1 minute (60 seconds) before the subthreshold dose of collagen. (Controls not shown demonstrated the stimulatory effect of GSH (10 μM) or GSH (10 μM) with GSSG [2 μM], added simultaneously with the same dose of collagen.) These experiments were performed a minimum of 2 times with similar results. (C) Platelets were incubated without GSH (lane 1), with GSH (10 μM; lane 2), or with GSSG (5 μM; lane 3) for 5 minutes at 24° C under nonstirring conditions. The sulfhydryl reagent 3-N-maleimidylpropionyl biotin (MPB; 50 μM) was added in excess of the GSH, and the labeling performed under conditions that cause disruption of the αIIbβ3 receptor (5 mM EDTA [ethylenediaminetetraacetic acid], 60 minutes, 37° C). Equal amounts of protein were added to lanes 1, 2, and 3. (D) Densitometry of the individual bands was used to obtain quantitative results. The first 2 columns show the ratio of labeling of the β3 subunit with GSH or GSSG to the β3 subunit without GSH (± 1 SE, n = 8). As a control for possible translocation of αIIbβ3 the third and fourth columns give the ratio of labeling using a label for primary amines, sulfosuccinimido-biotin (SSB) in the β3 subunit with GSH or GSSG to the β3 subunit without GSH (± 1 SE).
