Fig 2: 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] EC50 was not significantly different (95% CI of WT EC50: 0.042 to 0.090, 95% CI of Stx8-/- EC50: 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 aIIbß3 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 µm exogenous ADP response to EC50 concentration of thrombin (i) and CRP (ii). D, aggregation in response to co-stimulation with ~EC50 (0.05–0.075 units/ml) thrombin and 10 µm ADP was compared with EC50 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 EC50 CRP (0.3–0.5 µg/ml) was measured (n = 4). There was a significant reduction in aggregation in response to ~EC50 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 EC50 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.

Fig 3: Analysis of secretion in Vti1b-/- platelets. A, expression levels of other known SNAREs and SNARE-associated proteins were measured in Stx8-/- platelets to ensure the defect results from Stx8 deletion. Levels of STX11, VAMP8, SNAP23, and MUNC13d and 18b were comparable between the genotypes (image representative of at least 3 independent experiments). B, a significant decrease in expression of Vti1b in Stx8-/- platelets was observed (31.13 ± 5.85% of WT expression, p < 0.01). C, similarly, Stx8 was also down-regulated in Vti1b-/- platelets. D, to confirm that only complete ablation of Stx8 leads to defective dense granule secretion, we analyzed ATP secretion from Stx8+/- platelets and found no defect, unlike in Stx8-/- platelets (n > 3, difference not significant but consistent for WT versus Stx8-/-, two-way ANOVA). E–G, to further confirm that deletion of Stx8 and not the associated down-regulation of Vti1b was responsible for defect in dense granule secretion observed, we measured secretion from dense (E) and a-granules (F) and lysosomes (G) in Vti1b-/- platelets. No difference was observed in any of the secretion events in Vti1b-/- platelets (n = 6). Error bars represent S.E.

Fig 4: Expression of SNARE STX8 in platelets, its localization and interactions. A, resting human and mouse platelets were lysed in SDS sample buffer and proteins were separated on SDS-PAGE. The resulting blots were probed with antibodies as shown. B, human double washed platelets were solubilized using digitonin as described. The resulting digitonin-soluble (cytosol) and digitonin-insoluble (membrane) fractions were separated on SDS-PAGE and blotted as before and localization of STX8 was compared with localization of Fc receptor ? signaling chain (FcR?, membrane marker), GAPDH (cytosol marker), and STX11 (membrane-associated SNARE). C, human resting (R) and activated (A, 1 unit/ml of thrombin) double washed platelets were lysed as described and proteins were immunoprecipitated (IP) with relevant antibodies. The resultant immune complexes were denatured, separated on SDS-PAGE, and blotted as before. Rabbit nonspecific Ig controls (same species as IP antibodies) were also included. D, the STX8-STX11 complex identified was found to be constitutive, with no significant change upon activation as quantified using ImageJ (n = 4). Data shown (A–C) are representative of at least 3 experiments, error bars represent S.E.

Fig 5: Initial characterization of Stx8-/- platelets. A, resting platelets were prepared for TEM imaging, and images were acquired using Tecnai-12 electron microscope at ×4300 magnification. Arrows denote a granules, arrowheads denote dense granules (scale bar: 1 µm). Images are representative of 3 independent observations. B, the levels of glycoprotein expression were measured using FACS and compared with expression levels of WT platelets. There was no significant difference between the genotypes (n = 5, p > 0.05, two-way ANOVA). Error bars represent S.E.