Journal: The Journal of Biological Chemistry
Article Title: Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability *
doi: 10.1074/jbc.M114.602615
Figure Lengend Snippet: Analysis of aggregation and ADP rescue experiments in Stx8 −/− platelets. A, aggregation was measured using lumi-aggregometer and amplitude was expressed as percentage decrease in optical density of the sample at t = 180 s ( n = 4). The concentration-response curve for Stx8 −/− was shifted to the right, but [thrombin] EC 50 was not significantly different (95% CI of WT EC 50 : 0.042 to 0.090, 95% CI of Stx8 −/− EC 50 : 0.069 to 0.101, p > 0.05). However, the maximum aggregation amplitude was significantly reduced in Stx8 −/− at 0.075 units/ml of thrombin (26.1 ± 13.6% decrease in optical density in Stx8 −/− versus 60.8 ± 18.2% in WT, p = 0.021, two-way ANOVA with Bonferroni post-test), with aggregation at 0.05 and 0.06 units/ml also consistently reduced, but not significantly. B, integrin α IIb β 3 activation was measured by FACS, no difference in PE-JON/A binding was observed between genotypes suggesting an alternative mechanism for the observed aggregation defect. C, representative aggregation traces showing the effect of co-stimulation with 10 μ m exogenous ADP response to EC 50 concentration of thrombin ( i ) and CRP ( ii ). D, aggregation in response to co-stimulation with ∼EC 50 (0.05–0.075 units/ml) thrombin and 10 μ m ADP was compared with EC 50 thrombin alone ( n ≥ 5). Stx8 −/− aggregation was significantly reduced in response to thrombin alone (37.8 ± 8.9 versus 63.4 ± 6.1% decrease in optical density, respectively, p < 0.05) and could be fully rescued with ADP ( p > 0.05). The maximum aggregation to 1 unit/ml of thrombin was the same between genotypes (data not shown). Similarly, the effect of co-stimulation with ADP and EC 50 CRP (0.3–0.5 μg/ml) was measured ( n ≥ 4). There was a significant reduction in aggregation in response to ∼EC 50 CRP in Stx8 −/− platelets (30.0 ± 7.3 versus 46.3 ± 8.6% in WT, p < 0.05) that was fully rescued with ADP ( p > 0.05). Maximum response (to 5 μg/ml of CRP) was the same (data not shown). E, secretion of ATP was also measured. Again, secretion in response to EC 50 concentrations of thrombin or CRP was significantly reduced ( p < 0.01), whereas co-stimulation with ADP could only partially rescue secretion when thrombin was used as primary agonist ( p < 0.05). In the case of co-stimulation with CRP, ADP enhanced ATP secretion to a much greater extent in both WT and Stx8 −/− platelets (ADP enhancement of 185.75 ± 42.55% of CRP alone, compared with 111.30 ± 20.66% of thrombin alone). Error bars represent S.E.
Article Snippet: Despite using four different STX8 antibodies in this project (Synaptic Systems polyclonal rabbit anti-STX8 (number 110-083), Santa Cruz Biotechnology monoclonal mouse anti-STX8 ( ) (sc-136092), in-house rabbit polyclonal anti-STX8 antibody developed at the University of Bielefeld, and R&D Systems sheep polyclonal anti-STX8 antibody (AF5448)) we did not manage to immunoprecipitate STX8 in this project to provide the reciprocal control (data not shown).
Techniques: Concentration Assay, Activation Assay, Binding Assay
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![Quantification of granule secretion in Stx8 −/− platelets. All concentration-response data were analyzed by appropriate curve fitting in GraphPad Prism. A, mouse platelets were stimulated in the aggregometer as described. Dense granule secretion was measured as an increase in luminescence relative to ATP standard for each stimulation. Data were normalized to maximum ATP secretion of WT platelets on the day of experiment. There was a significant increase in [thrombin] EC 50 in Stx8 −/− (95% CI of EC 50 : 0.085 to 0.11 units/ml) compared with WT platelets (95% CI of EC 50 : 0.068 to 0.086 units/ml) ( n = 5, variable slope sigmoidal dose-response curve, p = 0.0248). Stx8 −/− and WT secretion in response to 0.06 units/ml was, respectively, 1.742 ± 0.618 and 10.379 ± 3.767% of the maximum WT secretion on the day ( n = 4, p > 0.05, ns), and in response to 0.075 units/ml of thrombin: 6.927 ± 5.970 and 38.933 ± 20.969% of the maximum WT secretion, p = 0.0339). B, the total 5-HT content was measured using 3 H-labeled 5-HT as described. There was no significant difference in 5-HT content between genotypes ( n = 3). C, α-granule secretion was measured by FACS. Median fluorescent intensity (MFI) of FITC-CD62P was quantified in response to increasing concentrations of thrombin. MFI was normalized to the maximum MFI of WT platelets on the day of experiment ( n = 4). D, lysosome secretion was measured by β-hexosaminidase enzyme activity in supernatants following stimulation with increasing concentrations of thrombin. Absorbance at 405 nm was measured, and values were expressed as percentage of TOTAL control for each subject ( n = 6). There was no difference in [thrombin] EC 50 between genotypes in α-granule or lysosome secretion ( p > 0.05, variable slope sigmoidal dose-response curve). Error bars represent S.E.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0400/pmc04340400/pmc04340400__zbc0051505600003.jpg)
![Analysis of aggregation and ADP rescue experiments in Stx8 −/− platelets. A, aggregation was measured using lumi-aggregometer and amplitude was expressed as percentage decrease in optical density of the sample at t = 180 s ( n = 4). The concentration-response curve for Stx8 −/− was shifted to the right, but [thrombin] EC 50 was not significantly different (95% CI of WT EC 50 : 0.042 to 0.090, 95% CI of Stx8 −/− EC 50 : 0.069 to 0.101, p > 0.05). However, the maximum aggregation amplitude was significantly reduced in Stx8 −/− at 0.075 units/ml of thrombin (26.1 ± 13.6% decrease in optical density in Stx8 −/− versus 60.8 ± 18.2% in WT, p = 0.021, two-way ANOVA with Bonferroni post-test), with aggregation at 0.05 and 0.06 units/ml also consistently reduced, but not significantly. B, integrin α IIb β 3 activation was measured by FACS, no difference in PE-JON/A binding was observed between genotypes suggesting an alternative mechanism for the observed aggregation defect. C, representative aggregation traces showing the effect of co-stimulation with 10 μ m exogenous ADP response to EC 50 concentration of thrombin ( i ) and CRP ( ii ). D, aggregation in response to co-stimulation with ∼EC 50 (0.05–0.075 units/ml) thrombin and 10 μ m ADP was compared with EC 50 thrombin alone ( n ≥ 5). Stx8 −/− aggregation was significantly reduced in response to thrombin alone (37.8 ± 8.9 versus 63.4 ± 6.1% decrease in optical density, respectively, p < 0.05) and could be fully rescued with ADP ( p > 0.05). The maximum aggregation to 1 unit/ml of thrombin was the same between genotypes (data not shown). Similarly, the effect of co-stimulation with ADP and EC 50 CRP (0.3–0.5 μg/ml) was measured ( n ≥ 4). There was a significant reduction in aggregation in response to ∼EC 50 CRP in Stx8 −/− platelets (30.0 ± 7.3 versus 46.3 ± 8.6% in WT, p < 0.05) that was fully rescued with ADP ( p > 0.05). Maximum response (to 5 μg/ml of CRP) was the same (data not shown). E, secretion of ATP was also measured. Again, secretion in response to EC 50 concentrations of thrombin or CRP was significantly reduced ( p < 0.01), whereas co-stimulation with ADP could only partially rescue secretion when thrombin was used as primary agonist ( p < 0.05). In the case of co-stimulation with CRP, ADP enhanced ATP secretion to a much greater extent in both WT and Stx8 −/− platelets (ADP enhancement of 185.75 ± 42.55% of CRP alone, compared with 111.30 ± 20.66% of thrombin alone). Error bars represent S.E.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0400/pmc04340400/pmc04340400__zbc0051505600004.jpg)
