Fig 2: 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 3: 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] EC50 in Stx8-/- (95% CI of EC50: 0.085 to 0.11 units/ml) compared with WT platelets (95% CI of EC50: 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 3H-labeled 5-HT as described. There was no significant difference in 5-HT content between genotypes (n = 3). C, a-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 ß-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] EC50 between genotypes in a-granule or lysosome secretion (p > 0.05, variable slope sigmoidal dose-response curve). Error bars represent S.E.

Fig 4: In vivo thrombus formation and embolization of analysis in Stx8-/- and WT animals. Carotid artery damage was achieved by treatment with FeCl3 as described. Fluorescently labeled platelets adhering at the site of injury could then be imaged continuously. A, the rate of platelet adhesion was slower in Stx8-/-, but the difference was not significant (adhesion rate shown as mean ± S.E.). B, the final thrombi size (expressed as IFD of the field of view) at the end of a 20-min experiment was also reduced in Stx8-/- animals but the difference was not significant. The videos were then reviewed again, focusing on the number of embolization events as shown in C. An embolus, seen here as a small fragment of the shell of the growing thrombus (arrow at 0 s) becomes dislodged and initially “rolls” with the direction of blood flow (at 5 s) to eventually become detached from the thrombus and carried away by the blood flow (6 s). D, when counted throughout the duration of the experiment, the number of embolization events was significantly increased in the Stx8-/- (137.3 ± 20.12) compared with WT animals (75.46 ± 17.71) (n = 8, p = 0.037, t test). E, mean time to cessation of bleeding in tail bleeding assay was the same in Stx8-/- and WT animals (t < 180 s, n = 6). 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.