(A–C) Serial transplantation assay showed a significantly reduced contribution of Plaur^–/– Ly5.2⁺ donor cells to the hematopoietic system in secondary Ly5.1⁺ recipients. (A) Percentage of Ly5.2⁺ PBMCs in secondary Ly5.1⁺ recipient mice at 8 and 24 weeks after transplantation (Tx). (B) Number of CRUs per 5 × 10⁵ BMMCs in secondary Ly5.1⁺ recipient mice at 24 weeks after transplantation. (C) Multilineage repopulation in secondary Ly5.1⁺ recipient mice at 24 weeks after transplantation, revealing the percentage of GFP⁺ neutrophils (Gr1CD11b^hi; N), monocytes (Gr1CD11b^lo; M), B lymphocytes (B220⁺; B) and T lymphocytes (CD3e⁺; T). *P < 0.05 versus WT donor cells (n = 6–9).

(A) Decreased steady-state counts of various HSPC populations in the BM of Plaur^–/– compared with WT mice. *P < 0.05 versus WT (n = 4–9). (B) WT and Plaur^–/– mice were transplanted with syngeneic WT or Plaur^–/– BMCs after lethal irradiation. At 6 weeks after transplantation, peripheral blood counts in the recipient mice were fully normalized (not shown). Counting of the number of CFU-Cs in the BM revealed that the number of HSPCs was reduced in the BM of mice grafted with Plaur^–/– BM. Consistent herewith, fewer CFU-Cs were detected in nontransplanted Plaur^–/– mice (not shown). *P < 0.05 (n = 4). (C) Fewer lethally irradiated splenectomized WT mice survived when transplanted with 1 × 10⁵ Plaur^–/– BMCs compared with WT BMC transplantation. P < 0.05 versus WT, Cox regression analysis (n = 17).

(A and B) Representative FACS histogram plots of cell-cycle analysis of ^MuPAR⁺ (A) and ^MuPAR^– (B) Lin^–cKit⁺ HSPCs in steady-state conditions. (C) Quantitative analysis of the cell-cycle status in Lin^–cKit⁺ HSPCs in steady-state conditions. Compared with ^MuPAR⁺ HSPCs, fewer HSPCs in the ^MuPAR^– fraction were in G₀/G₁. *P < 0.05 (n = 4). (D–F) Compared with ^MuPAR⁺ HSPCs, fewer Lin^–cKit⁺ HSPCs in the ^MuPAR^– fraction were Pyronin Y^lo (D), while more cells proliferated (E) or were apoptotic (F). *P < 0.05 (n = 4). (G) Hematopoietic recovery in WT and Plaur^–/– mice after 5-FU treatment. Compared with WT mice, the wbc counts in Plaur^–/– mice were lower at 7, 11, and 14 days after administration of 250 mg/kg 5-FU. P < 0.05 versus WT (ANOVA); *P < 0.05 versus respective WT (n = 11). (H) Survival of Plaur^–/– mice after treatment with 250 mg/kg 5-FU i.v. was reduced compared with WT mice. P < 0.05 versus WT, Cox regression analysis (n = 11).

(A and B) Fewer circulating CFU-Cs (A) and CFU-Ss (B) were observed in Plg^–/– mice 5 days after G-CSF treatment, but not in steady-state conditions. *P < 0.05 versus WT (n = 8–15). (C) Fewer lethally irradiated syngeneic WT recipients survived after transplanting 1 × 10⁵ PBMCs from G-CSF–treated Plg^–/– mice than from WT mice. P < 0.05, Cox regression analysis (n = 20–39). (D) The median fluorescence intensity (MFI) BR4 signal, which specifically recognizes only intact ^MuPAR (see Methods), on Sca-1⁺ BMCs was reduced 2 days after 5-FU treatment in WT but not Plg^–/– mice. ^#P < 0.05 versus baseline (n = 4). (E and F) Fewer circulating CFU-Cs (E) and CFU-Ss (F) were observed in Plaur^–/– mice 5 days after G-CSF treatment, but not in steady-state conditions. *P < 0.05 versus WT (n = 11–15). (G and H) Compared with controls, fewer CFU-Cs (G; n = 12–18) and CFU-Ss (H; n = 10) were mobilized after G-CSF treatment in Plaur^–/– or WT recipients of Plaur^–/– BM transplant. Transplantation of WT BM into Plaur^–/– recipients completely restored CFU-C and CFU-S mobilization after G-CSF treatment. *P < 0.05.

(A) Homing of BMCs in lethally irradiated nonsplenectomized recipient mice. Compared with control IgG, pretreatment with 2 μg anti-^MuPAR per 10⁶ cells reduced the number of GFP⁺ BMCs homing to the BM, but not to the spleen, 5 days after transplantation of 5 × 10⁶ donor cells. *P < 0.05 versus control IgG (n = 4). (B) Homing and early engraftment of Ly5.1⁺Lin^–cKit⁺ HSPCs to the BM of lethally irradiated splenectomized Ly5.2⁺ recipient mice. Compared with control IgG, pretreatment with anti-^MuPAR inhibited the homing and early engraftment of HSPCs to the BM 5 days after transplantation. *P < 0.05 versus control IgG (n = 5–6). (C–E) Adhesion of 5 × 10⁴ WT Lin^–cKit⁺ HSPCs to a monolayer of OP9 mouse BM stromal cells (C), sVCAM-1 (D), or fibronectin (E). Compared with control IgG, pretreatment with anti-^MuPAR inhibited HSPC adhesion. *P < 0.05 versus control IgG (n = 8). (F) Compared with control IgG, fewer lethally irradiated splenectomized WT mice survived when transplanted with 1 × 10⁵ WT BMCs pretreated with anti-^MuPAR, indicating that the expression of ^MuPAR on HSPCs promotes engraftment. P < 0.05 versus control (Cox regression; n = 10).

(A–C) Double-fluorescent immunostaining of a longitudinal section through the femur, followed by nuclear DAPI staining (blue), revealed the expression of α₄β₁ (green; A) and ^MuPAR (red; B) near the endosteal bone. The merged image (C) shows coexpression of α₄β₁ and ^MuPAR on the same cells near the endosteal bone (yellow). Dashed line demarcates the border between cortical bone (B) and BM; dotted line distinguishes the bone (left) from the bone marrow cavity (right). Scale bars: 10 μm. (D) Homing and early engraftment of Ly5.1⁺ Lin^–cKit⁺ HSPCs to the BM of lethally irradiated splenectomized Ly5.2⁺ recipient mice. Compared with control IgG, pretreatment with 2 μg anti-α₄β₁ per 10⁶ cells inhibited the homing and early engraftment of HSPCs to the BM 5 days after transplantation. Anti-^MuPAR did not further reduce homing and early engraftment when α₄β₁ was inhibited, which suggests that ^MuPAR and α₄β₁ act in the same pathway. Data with anti-^MuPAR from Figure 3B are repeated for comparison. *P < 0.05 versus control IgG (n = 5–6). (E–G) Adhesion of 5 × 10⁴ WT Lin^–cKit⁺ HSPCs to OP9 mouse BM stromal cells (E), sVCAM-1 (F), or fibronectin (G). Compared with control IgG, pretreatment with anti-α₄β₁ inhibited HSPC adhesion. Anti-^MuPAR did not further reduce adhesion when α₄β₁ was inhibited, which suggests that ^MuPAR and α₄β₁ act in the same pathway. Data with anti-^MuPAR from Figure 3, C–E, are repeated for comparison. *P < 0.05 versus control IgG (n = 8).