Characterization of Vps33b^fl/fl-ER^T2 platelet function. (A) VWF and P-selectin levels were measured in platelets by immunoblot analysis. β-Actin was used as a loading control. Densitometric analysis showed a reduction in VWF and P-selectin content in Vps33b^fl/fl-ER^T2 platelets (n = 3 mice) in comparison with Vps33b^fl/fl platelets (n = 5 mice). (B) Thrombin (0.1 U/mL) induced P-selectin expression on the surface of Vps33b^fl/fl-ER^T2 (n = 15 mice) platelets in comparison with Vps33b^fl/fl (n = 19 mice). Histogram (left) shows an IgG control (purple), P-selectin expression in controls (green and pink), and Vps33b^fl/fl-ER^T2 platelets (blue). (C) Representative aggregation responses to thrombin (0.05 U/mL), collagen (3 μg/mL), and ADP (3 μM) were similar in washed platelets from control and Vps33b^fl/fl-ER^T2 mice (n = 12 mice per genotype). (D) δ-Granule secretion was measured in the lumi-aggregometer using a luciferase assay (ATP). ATP secretion in Vps33b^fl/fl and Vps33b^fl/fl-ER^T2 platelets (n = 12 mice per genotype) in response to 0.05 U/mL thrombin. (E) Reduced ATP secretion was observed in Vps33b^fl/fl-ER^T2 vs Vps33b^fl/fl platelets (n = 12 mice per genotype) upon collagen stimulation. (F) ATP secretion measured by lummi-aggregometry after stimulation of washed platelets with the divalent calcium ionophore A23187 (10 μM) (n = 5 mice per genotype). (G) Tail-bleeding assay (n = 15 to 17 mice per genotype). Open circles (○) represent individual mice. Horizontal lines represent means. (H) Representative fluorescence images (DiOC₆) at 2 and 4 minutes of blood perfusion (left and middle panels). Representative phase-contrast images (BF) at the end of the perfusion period (right panel). Images were obtained using a Zeiss Axiovert 200 inverted high-end microscope with a 40× objective. Scale bar, 10 µm. All values are mean ± SEM. *P < .05; **P < .01; ***P < .001. BF, bright field; MFI, mean fluorescence intensity; ns, not significant.