Warfarin impairs hematopoiesis via periostin in the BMM. (A) Western blot of protein lysates of macrophages, the stroma cell line MS-5, MSCs, and the osteoblast precursor cell line MC3T3 (as a known producer of periostin³³ ) stained with an antibody to periostin (90 kDa). Vertical lines have been inserted to indicate a repositioned gel lane. The blot is representative of 3 independent western blots. (B) Co-immunoprecipitation of concentrated CM from vehicle- or warfarin-treated macrophages with an anti-Gla antibody. The western blot was performed with an antibody to periostin (90 kDa). The upper band represents periostin at 90 kDa, marked by an arrow. The blot is representative of 3 independent experiments. (C) Total number of colonies formed by 10 000 BM cells from mice treated with vehicle (red circles), warfarin (blue squares), or warfarin plus periostin (green triangles) in methylcellulose assays (P = .01, ANOVA, Tukey Test, n = 6-9). As seen in the schematic for panel D, 4 μg/day periostin had been added on days 10 to 14 of the warfarin treatment. (D) Percentage of CD11b⁺ myeloid cells in the peripheral blood of control mice (red circles), control mice treated with BSA (blue squares), or mice treated with warfarin (green triangles) or warfarin plus periostin (purple inverted triangles). The increase of the percentage of myeloid cells after a 5-day treatment with periostin is significant (P = .05; ANOVA, Tukey test, n = 5). Periostin was administered at 4 μg/d from day 10 to 14 of concomitant warfarin treatment. (E) Total number of leukocytes 7 days after a 1-time treatment with 200 mg/kg 5-FU in mice treated with vehicle (red circles) or mice treated with 0.5 mg/kg warfarin (blue squares) or warfarin plus periostin (green triangles; P = .03; ANOVA, Tukey Test, n = 6-10). The schematic details the treatment schedule. (F) Ratio of CD45.1/CD45.2 in peripheral blood 4 and 8 weeks after competitive transplantation of 1 × 10⁶ CD45.1⁺ total donor BM from vehicle-treated (red circles), warfarin-treated (blue squares), or warfarin-treated plus periostin (green triangles) mice, mixed with 10⁶ CD45.2⁺ competitor cells and transplanted into CD45.2⁺ recipient mice (ANOVA, Tukey Test, n = 8). (G) Ratio of CD45.1/CD45.2 4 weeks after competitive transplantation of 10⁶ CD45.1⁺ total donor BM from vehicle-treated (red circles) or periostin-treated (blue squares) mice (no warfarin treatment), mixed with 10⁶ CD45.2⁺ competitor cells and transplanted into CD45.2⁺ recipient mice (P = .02; t test, n = 10).