Figure 1.
Figure 1. MK S1P, S1P1S1P, and the S1P gradient are dispensable for platelet production in mice. (A) Current literature suggests that S1P supports platelet production by 2 independent mechanisms: S1P1 senses the S1P gradient to promote PP extensions toward blood sinusoids (purple) and further supports fragmentation, and S1P supports platelet fragmentation by receptor-independent promotion of Src family kinase (SFK) expression and activation in MKs (orange). Removal of the S1P gradient and MK S1P production would thus be predicted to have cumulative effects on platelet production. Target cells of Cre alleles used in this study are indicated. (B) Peripheral blood platelet and lymphocyte counts in mice with combined loss of lymphatic endothelium and hematopoietic S1P production (Sphk1f/−:2f/−:Mx1Cre+) and with alternative gradient ablation by impaired S1P breakdown (Sgpl1−/). (C) Relative changes in the same cell populations after supplying the functional S1P1 antagonist fingolimod (2 mg/L) in the drinking water of wild-type mice for 1 week. (D) Platelet and lymphocyte counts in mice after deletion of S1pr1 in hematopoietic and other cells (postnatal induction, Mx1Cre+), in all hematopoietic cells (constitutive deletion, Vav1 Cre+), in MKs (constitutive deletion; Pf4Cre+), or endothelial cells and MKs (postnatal induction, PdgfbiCreERT2+). (E-F) Platelet and lymphocyte counts in adult S1pr1f/f:Mx1Cre+/− mice 1 month after 3 consecutive injections of Poly IC (E) or in lethally irradiated wild-type mice 1 month after transfusion of S1pr1f/f:Mx1Cre+/− BM cells (F). (G) Relative change in platelet counts 24 hours after injections of the S1P1 agonist SEW2871 or antagonist W146 or respective vehicle controls, as indicated (W146 was injected either as a single bolus [middle] or at 0, 6, 12, and 18 hours [right]). Lymphocyte counts and acute effects of drug treatment in supplemental Figure 1. (H) Platelet half-life, mean platelet volume (MPV), and plasma S1P levels in S1pr1f/f:Mx1Cre+ mice. (I) RBC counts in mice with hematopoietic deletion of S1pr1 or Sphk1&2 (the same mice as in Figure 1D, 1F, 1E, and 1B, respectively). (J) Representative microcomputed tomography images of femurs from S1pr1f/f:Mx1Cre+ mice and littermate controls. Representative coronal and transverse sections (approximate area indicated) are shown, quantification in supplemental Figure 2. (K) BM progenitors as percentage of total bone marrow cells and MK density in BM and spleen of S1pr1f/f:Mx1Cre+ mice. Representative images in supplemental Figure 3. All animals are compared with their respective littermate controls, n indicates the number of animals from which samples were obtained, mean + standard error of the mean shown. Statistical analyses by Mann-Whitney U test. ns, not significant.

MK S1P, S1P1S1P, and the S1P gradient are dispensable for platelet production in mice. (A) Current literature suggests that S1P supports platelet production by 2 independent mechanisms: S1P1 senses the S1P gradient to promote PP extensions toward blood sinusoids (purple) and further supports fragmentation, and S1P supports platelet fragmentation by receptor-independent promotion of Src family kinase (SFK) expression and activation in MKs (orange). Removal of the S1P gradient and MK S1P production would thus be predicted to have cumulative effects on platelet production. Target cells of Cre alleles used in this study are indicated. (B) Peripheral blood platelet and lymphocyte counts in mice with combined loss of lymphatic endothelium and hematopoietic S1P production (Sphk1f/−:2f/−:Mx1Cre+) and with alternative gradient ablation by impaired S1P breakdown (Sgpl1−/). (C) Relative changes in the same cell populations after supplying the functional S1P1 antagonist fingolimod (2 mg/L) in the drinking water of wild-type mice for 1 week. (D) Platelet and lymphocyte counts in mice after deletion of S1pr1 in hematopoietic and other cells (postnatal induction, Mx1Cre+), in all hematopoietic cells (constitutive deletion, Vav1 Cre+), in MKs (constitutive deletion; Pf4Cre+), or endothelial cells and MKs (postnatal induction, PdgfbiCreERT2+). (E-F) Platelet and lymphocyte counts in adult S1pr1f/f:Mx1Cre+/− mice 1 month after 3 consecutive injections of Poly IC (E) or in lethally irradiated wild-type mice 1 month after transfusion of S1pr1f/f:Mx1Cre+/− BM cells (F). (G) Relative change in platelet counts 24 hours after injections of the S1P1 agonist SEW2871 or antagonist W146 or respective vehicle controls, as indicated (W146 was injected either as a single bolus [middle] or at 0, 6, 12, and 18 hours [right]). Lymphocyte counts and acute effects of drug treatment in supplemental Figure 1. (H) Platelet half-life, mean platelet volume (MPV), and plasma S1P levels in S1pr1f/f:Mx1Cre+ mice. (I) RBC counts in mice with hematopoietic deletion of S1pr1 or Sphk1&2 (the same mice as in Figure 1D, 1F, 1E, and 1B, respectively). (J) Representative microcomputed tomography images of femurs from S1pr1f/f:Mx1Cre+ mice and littermate controls. Representative coronal and transverse sections (approximate area indicated) are shown, quantification in supplemental Figure 2. (K) BM progenitors as percentage of total bone marrow cells and MK density in BM and spleen of S1pr1f/f:Mx1Cre+ mice. Representative images in supplemental Figure 3. All animals are compared with their respective littermate controls, n indicates the number of animals from which samples were obtained, mean + standard error of the mean shown. Statistical analyses by Mann-Whitney U test. ns, not significant.

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