Etv6^R355X is homozygous lethal and heterozygotes display abnormal platelets and increased B cells. (A) Schematic diagram depicting the functional domains of ETV6 and location of the ETV6:p.R359X variant. (B) Genotypes generated by Etv6^R355X/+ × Etv6^R355X/+ mating, showing no Etv6^R355X/R355X pups. (C) Western blot and quantification of ETV6 protein levels in B220-enriched splenocytes from Etv6^+/+ and Etv6^R355X/+ mice as well as 293 T cells transfected with empty vector, wild-type Etv6, or Etv6^R355X. Etv6 contains an alternative initiation site at M43 resulting in 2 bands visible by western blot. (D) Peripheral blood platelet count in Etv6^+/+ (n = 12) and Etv6^R355X/+ (n = 11) mice. Data are from 2 combined experiments and show mean ± standard deviation (SD). (E) Representative images and quantification of ex vivo clot retraction using platelets from Etv6^+/+ (n = 4) and Etv6^R355X/+ (n = 4) mice, showing percent of initial clot area at 0, 1, and 2 hours. Data are representative of 2 experiments. (F) Transmission electron microscopy images of platelets from Etv6^+/+ and Etv6^R355X/+ mice and quantification of platelet area from Etv6^+/+ (n = 192) platelets and Etv6^R355X/+ (n = 92) platelets. (G) Representative gating strategy to identify leukocyte populations, showing representative plots from Etv6^+/+ (top) and Etv6^R355X/+ (bottom) mouse spleens. All populations are gated directly off live singlets. Gr1 labels both granulocytes and monocytes, gates are set using a tissue with a visible Gr1⁺ population (ie, BM; supplemental Figure 1B) and applied to all samples. (H) Frequency (top) and absolute number (bottom) of splenic leukocytes in Etv6^+/+ (green, n = 18) and Etv6^R355X/+ (red, n = 17) mice at 3 months of age. Data are from 5 combined experiments and show mean ± SD. ∗P < .05; ∗∗P < .001; and ∗∗∗P < .0001, as determined by unpaired t tests or two-way analysis of variance (ANOVA) with a Holm-Šidák multiple comparisons test.