Cell Biology

Synergistic effects of deleting the tyrosine phosphatases Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis in mice

Elsa Barré
Marc-Damien Lourenco-Rodrigues
Lucie Zimmermann
Marion Pugliano
Cécile Loubière
Fabienne Proamer
Jean-Yves Rinckel
Anita Eckly
Zihan Qu
Jinmin Miao
Zhong-Yin Zhang
Yotis A Senis
Alexandra Mazharian author has email address

Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche - S 1255, Etablissement Français du Sang Grand Est, Université de Strasbourg, Strasbourg, France
James Tarpo Jr. and Margaret Tarpo Department of Chemistry, Purdue University, West Lafayette, United States
Borch Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, United States
Institute for Drug Discovery, Purdue University, West Lafayette, United States

https://doi.org/10.7554/eLife.110053.1

Open access
Copyright information

Figures and data

Protein expression of Shp1 and Shp2 in megakaryocytes and platelets from Gp1ba-Cre mice.
(i) Representative blots of Shp1 and Shp2 expression levels from CT, Shp1, Shp2 and Shp1/2 DKO mice by capillary immunoassays with the respective antibodies in (A) sorted-MK progenitors and (B) washed platelets. Percentage of residual level of (ii) Shp1 and (iii) Shp2 in Shp1 KO (blue), Shp2 KO (green) and Shp1/2 DKO (red) mice. n = 3-6 mice per genotype. Mean ± SEM; one way-ANOVA; ** P < 0.01, *** P < 0.001.

Macrothrombocytopenia and mild increased bleeding in Shp1/2 DKO mice.
(A) Platelet counts (i) and platelet volumes (ii) of control (CT) (n=75), Shp1 KO (n=21), Shp2 KO (n=29) and Shp1/2 DKO (n=46) mice. Mean ± SEM; one way-ANOVA; *** P < 0.001. (B) (i) Cumulative bleeding time was recorded (ii) and the volume of blood loss was measured over 30 minutes. Mean ± SEM; one way-ANOVA; * P < 0.05. (C) Mean fluorescence intensity (MFI) of platelet (i) GPVI and (ii) α2 integrin expression in CT (n=10), Shp1 KO (n=4), Shp2 KO (n=4) and Shp1/2 DKO (n=13) mice. Mean ± SEM; one way-ANOVA; *** P < 0.001.

Aberrant platelet function in Shp1/2 DKO mice.
(A-C) Aggregation of washed platelets were measured by lumi-aggregometry in response to agonists indicated. Representative traces, n= 4-8 mice/genotype per condition, percentage of aggregation at 5 minutes and area under the curve (AUC) quantification. Mean ± SEM, n = 5-17 mice/genotype per condition, one way-ANOVA, * P < 0.05. (D) Mean fluorescence intensity (MFI) of P-selectin expression of control (CT), Shp1 KO, Shp2 KO and Shp1/2 DKO platelets in whole blood in response to (i) 1 and 3 μg/mL CRP and (ii) 100 and 500 μM PAR4 peptide (PAR4p). Mean ± SEM, n = 5-9 mice/genotype per condition, two way-ANOVA; * P < 0.05, *** P < 0.001.

Aberrant platelet function in Shp1/2 DKO mice.
(A-C) Aggregation of washed platelets were measured by lumi-aggregometry in response to agonists indicated. Representative traces, n= 4-8 mice/genotype per condition, percentage of aggregation at 5 minutes and area under the curve (AUC) quantification. Mean ± SEM, n = 5-17 mice/genotype per condition, one way-ANOVA, * P < 0.05. (D) Mean fluorescence intensity (MFI) of P-selectin expression of control (CT), Shp1 KO, Shp2 KO and Shp1/2 DKO platelets in whole blood in response to (i) 1 and 3 μg/mL CRP and (ii) 100 and 500 μM PAR4 peptide (PAR4p). Mean ± SEM, n = 5-9 mice/genotype per condition, two way-ANOVA; * P < 0.05, *** P < 0.001.

Aberrant platelet signaling in Shp1/2 DKO mice.
Whole cell lysates of resting and (A) 10 µg/ml CRP-stimulated and (B) 10 µg/ml activating CLEC-2 antibody platelets from Shp1 KO, Shp2 KO and Shp1/2 DKO mice and litter-matched CT mice were western blotted with anti-Src p-Ty418, Src total, -Syk p-Tyr519/520 and Syk antibodies. (i) Representative blots of capillary-based immunoassays and (ii) quantification of peak areas from three independent experiments, Mean ± SEM, n = 3-4 mice/genotype; one-way ANOVA, *** P < 0.001.

Aberrant platelet signaling in Shp1/2 DKO mice.
Whole cell lysates of resting and (A) 10 µg/ml CRP-stimulated and (B) 10 µg/ml activating CLEC-2 antibody platelets from Shp1 KO, Shp2 KO and Shp1/2 DKO mice and litter-matched CT mice were western blotted with anti-Src p-Ty418, Src total, -Syk p-Tyr519/520 and Syk antibodies. (i) Representative blots of capillary-based immunoassays and (ii) quantification of peak areas from three independent experiments, Mean ± SEM, n = 3-4 mice/genotype; one-way ANOVA, *** P < 0.001.

Defects in megakaryocyte maturation and Tpo signaling in Shp1/2 DKO.
(A) Mature BM-derived MKs from Shp1 KO, Shp2 KO, Shp1/2 DKO, and litter-matched CT mice were stained with propidium iodide and ploidy of cells was quantified by flow cytometry. (i-ii) The percentage of 2-4N and 8-128N ploidy cells was quantified (n = 4-6 mice/genotype; mean ± SEM; two-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001). (B) Ex vivo proplatelet formation. Percentage of MKs forming proplatelet was quantified in culture. Mean ± SEM, two-way ANOVA, *** P < 0.001. (C) Mature BM-derived MKs from Shp1 KO, Shp2 KO, Shp1/2 DKO and litter-matched CT mice were stimulated with 50 ng/mL thrombopoietin (Tpo) for 10 min at 37°C. Whole cell lysates were western blotted with indicated antibodies. Representative blots capillary-based immunoassays and quantification of peak areas from n = 3 independent experiments/genotype; two-way ANOVA, *** P < 0.001.

Defects in megakaryocyte maturation and Tpo signaling in Shp1/2 DKO.
(A) Mature BM-derived MKs from Shp1 KO, Shp2 KO, Shp1/2 DKO, and litter-matched CT mice were stained with propidium iodide and ploidy of cells was quantified by flow cytometry. (i-ii) The percentage of 2-4N and 8-128N ploidy cells was quantified (n = 4-6 mice/genotype; mean ± SEM; two-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001). (B) Ex vivo proplatelet formation. Percentage of MKs forming proplatelet was quantified in culture. Mean ± SEM, two-way ANOVA, *** P < 0.001. (C) Mature BM-derived MKs from Shp1 KO, Shp2 KO, Shp1/2 DKO and litter-matched CT mice were stimulated with 50 ng/mL thrombopoietin (Tpo) for 10 min at 37°C. Whole cell lysates were western blotted with indicated antibodies. Representative blots capillary-based immunoassays and quantification of peak areas from n = 3 independent experiments/genotype; two-way ANOVA, *** P < 0.001.

Defects in ex vivo proplatelet formation and in vivo MK maturation in Shp1/2 DKO mice.
Bone marrow explants. Proportion of MKs from Shp1 KO, Shp2 KO, Shp1/2 DKO and litter-matched CT mice extending proplatelets at 6h of observation were observed. Bars represent the mean ± SEM of six independent experiments. (A) Quantification and (B) representatives’ images of round MKs, MKs with large ends and proplatelet (PPT) forming MKs. Scale bar: 50 µm. Mean ± SEM, one-way ANOVA, * P < 0.1, *** P < 0.001. (C) Classification of the MK according to their maturation stage: stage I (absence of granules), stage II (granules and developing demarcation membrane system (DMS) not yet organized), stage III (DMS organized in cytoplasmic territories). Data are reported as the percentage of the total number of MK, (i) of all stage MK and (ii) only stage 3 MK. Bars represent the mean ± SEM in three BM samples, *** P < 0.001. (D) Ploidy of in situ BM MKs measured by flow cytometry. Mean ± SEM, two-way ANOVA, * P < 0.1, *** P < 0.001.

Defects in ex vivo proplatelet formation and in vivo MK maturation in Shp1/2 DKO mice.
Bone marrow explants. Proportion of MKs from Shp1 KO, Shp2 KO, Shp1/2 DKO and litter-matched CT mice extending proplatelets at 6h of observation were observed. Bars represent the mean ± SEM of six independent experiments. (A) Quantification and (B) representatives’ images of round MKs, MKs with large ends and proplatelet (PPT) forming MKs. Scale bar: 50 µm. Mean ± SEM, one-way ANOVA, * P < 0.1, *** P < 0.001. (C) Classification of the MK according to their maturation stage: stage I (absence of granules), stage II (granules and developing demarcation membrane system (DMS) not yet organized), stage III (DMS organized in cytoplasmic territories). Data are reported as the percentage of the total number of MK, (i) of all stage MK and (ii) only stage 3 MK. Bars represent the mean ± SEM in three BM samples, *** P < 0.001. (D) Ploidy of in situ BM MKs measured by flow cytometry. Mean ± SEM, two-way ANOVA, * P < 0.1, *** P < 0.001.

Inhibition of Shp2 activity impairs megakaryopoiesis and Mpl signaling.
(A) Effects of two selective Shp1 allosteric inhibitors 10 µM M029 and 10 µM F2Ac were tested on megakaryopoeisis. (i) Viability, (ii) proliferation, and (iii) MK ploidy. Quantification from n = 3-4 independent experiments/condition. (B) Whole cell lysates of resting and 50 ng/mL Tpo-stimulated MKs were western blotted with the indicated antibodies. Representative blots capillary-based immunoassays and quantification of peak areas from n = 3 independent experiments/genotype. (C) Effects of two selective Shp2 allosteric inhibitors 10 µM SHP099 and 10 µM RMC-4550 were tested on megakaryopoeisis. (i) Viability, (ii) proliferation, (iii) MK ploidy and (iv) percentage of MKs forming proplatelets. Quantification from n = 3-6 independent experiments/condition. * P < 0.05; ** P < 0.01; *** P < 0.001, two-way ANOVA. (D) Whole cell lysates of resting and 50 ng/mL Tpo-stimulated MKs were western blotted with the indicated antibodies. Representative blots capillary-based immunoassays and quantification of peak areas from n = 3 independent experiments/genotype. * P < 0.05; ** P < 0.01; two-way ANOVA.

Inhibition of Shp2 activity impairs megakaryopoiesis and Mpl signaling.
(A) Effects of two selective Shp1 allosteric inhibitors 10 µM M029 and 10 µM F2Ac were tested on megakaryopoeisis. (i) Viability, (ii) proliferation, and (iii) MK ploidy. Quantification from n = 3-4 independent experiments/condition. (B) Whole cell lysates of resting and 50 ng/mL Tpo-stimulated MKs were western blotted with the indicated antibodies. Representative blots capillary-based immunoassays and quantification of peak areas from n = 3 independent experiments/genotype. (C) Effects of two selective Shp2 allosteric inhibitors 10 µM SHP099 and 10 µM RMC-4550 were tested on megakaryopoeisis. (i) Viability, (ii) proliferation, (iii) MK ploidy and (iv) percentage of MKs forming proplatelets. Quantification from n = 3-6 independent experiments/condition. * P < 0.05; ** P < 0.01; *** P < 0.001, two-way ANOVA. (D) Whole cell lysates of resting and 50 ng/mL Tpo-stimulated MKs were western blotted with the indicated antibodies. Representative blots capillary-based immunoassays and quantification of peak areas from n = 3 independent experiments/genotype. * P < 0.05; ** P < 0.01; two-way ANOVA.

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