Introduction

Platelets, or thrombocytes are small, anucleate blood cells derived from megakaryocytes (MKs) that are essential for hemostasis and thrombosis.¹ MK development, or megakaryopoiesis and platelet production, or thrombopoiesis are tightly regulated processes that take place in the bone marrow (BM), spleen and lungs, but the molecular mechanisms regulating these inter-related processes remain incompletely defined.^2,3 The primary driver of megakaryopoiesis, which encompasses differentiation of hematopoietic stem and progenitor cells (HSPCs) to MKs, proliferation, endomitosis and maturation is the cytokine thrombopoietin (Tpo) produced by the kidneys, liver and BM acting on its receptor, myeloproliferative leukemia protein (Mpl) expressed throughout the MK/platelet lineage.⁴ Tpo does not however explain which MKs undergo proplatelet formation and thrombopoiesis, which occurs when MKs come in contact with sinusoidal blood vessels. Several other cytokines and growth factors, extracellular matrix (ECM) proteins and chemokines are also required for optimal megakaryopoiesis and thrombopoiesis, as evidenced in mice lacking either Tpo or Mpl, which still produce MKs and platelets, albeit at much lower levels than normal mice.^5,6

The three primary signaling pathways downstream of Mpl are Janus kinase 2 (JAK2)/Signal transducer and activator of transcription (STAT), Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT, all of which culminate in nuclear translocation of signaling/transcription factors and gene regulation.⁷ Cytoskeletal remodeling via the RhoA/Rho-associated protein kinases (ROCK) pathway is also an integral part of megakaryopoiesis and thrombopoiesis, notably during endomitosis⁸, but the molecular link with the Mpl receptor is undefined. Clonal gain-of-function mutations in the Mpl/JAK2/STAT pathway in HSPCs are the most common causes of myeloproliferative neoplasms (MPN)⁹, characterized by increase MK/platelet production (essential thrombocythemia, ET), increased red blood cell production (polycythemia vera, PV) and associated fibrotic deposits (primary myelofibrosis, PMF).^10–12 Notably, the valine 617 phenylalanine (V617F) mutation in the pseudo-kinase domain of JAK2, resulting in increased catalytic active is the most prevalent, found in 60% of MPN cases, followed by C-terminal deletions in calreticulin, resulting in the release of truncated calreticulin and it acting as a ligand of Mpl in 30% of the cases, and mutations in Mpl resulting in increased expression in <5% of the cases.¹³ JAK2 and MAPK kinase (MEK) inhibitors are effective in some instances of MPN¹⁴, however there is a tendency of resistance and relapse, requiring alternative and improved therapeutics.

The Src homology 2 (SH2) domain-containing non-transmembrane protein-tyrosine phosphatases (PTPs) 1 and 2 (Shp1 and Shp2) are involved in hematopoietic cell differentiation, proliferation, function and survival, including megakaryopoiesis and thrombopoiesis.¹⁵ Intra- and inter-molecular interactions of the tandem SH2 domains with the catalytic domain and phospho-tyrosine binding partners regulate the activity and compartmentalization of both phosphatases, respectively. Tandem phospho-tyrosine residues in the C-terminal tails of Shp1 and Shp2 are implicated in substrate binding and regulation.¹⁶ Despite high structural similarities, the two phosphatases have distinct biological functions. Shp1, encoded by the PTPN6 gene, is primarily expressed in hematopoietic cells and is a negative regulator of immune receptor signaling, particularly the immunoreceptor tyrosine-based activation motif (ITAM)/Spleen tyrosine kinase (Syk)/phospholipase pathway.¹⁷ Shp2, encoded by the PTPN11 gene, is ubiquitously expressed and is a positive regulator of cytokine and growth factor signaling, specifically the Ras/MAPK and PI3K/AKT pathways.¹⁸ However, their functions go beyond these pathways and are context dependent. Downstream substrates and effectors of both phosphatases remain elusive.

We previously showed that targeted deletion of Shp1 and Shp2 in the MK/platelet lineage using the Pf4-Cre transgene/loxP system impairs megakaryopoiesis and thrombopoiesis in mice.¹⁵ However, we also found that these mice exhibited unexpected, non-MK/platelet-related defects, including a motheaten-like phenotype in Shp1-deficient mice, and severe myelofibrosis in mice lacking both phosphatases. Intriguingly, the motheaten-like phenotype was lost in Shp1/2 double-deficient mice, suggesting that Shp1 and Shp2 have opposing effect in this pathology. To determine whether these were in fact MK/platelet lineage-specific effects, we utilized the Gp1ba-Cre transgenic mouse to delete loxP-flanked Shp1 and Shp2 in mice. In contrast to Pf4-Cre transgenic mice, conditional deletion of Shp1 or Shp2 using the Gp1ba-Cre transgene had little if any detectable effect on megakaryopoiesis or thrombopoiesis in these mice under steady state conditions. However, deletion of both Shp1 and Shp2 using Gp1ba-Cre resulted in macrothrombocytopenia and aberrant MK development and maturation, due to a block in Mpl-mediated Ras/MAPK signaling. GPVI-induced platelet aggregation was also impaired due to reduced GPVI and α2 integrin subunit expression. Similar effects were seen with the structurally-distinct allosteric Shp2 inhibitors, SHP099 and RMC-4550.¹⁹ Findings demonstrate synergistic effects of deleting Shp1 and Shp2 in the MK/platelet lineage in mice by disrupting distinct signaling pathways, and highlight Shp2 as a potential therapeutic target in MPN.^20,21

Materials and Methods

Mouse models

All mice used were on a C57BL/6 background. Ptpn6^fl/fl, Ptpn11^fl/fl and Gp1ba-Cre^+/KI mice were generated, as previously described.^22–24 MK/Platelet specific Shp1 and Shp2 knockout (KO) mice were generated by crossing Ptpn6^fl/fl and Ptpn11^fl/fl mice with the Gp1ba-Cre transgenic deleter mouse Gp1ba-Cre^KI/+ mice. Gp1ba-Cre ^KI/+ were used as control mice (CT) (Supplemental Figure S1). All animal experiments were conducted in accordance with the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Permit Number: E67-482-10, Project approval number: APAFIS#28221-2020111714459066).

Inhibitors

SHP099 and RMC-4550 Shp2 allosteric inhibitors were purchased from MedChemExpress. M029 is a covalent allosteric Shp1 inhibitor (Z.Q. and Z.Y.Z, manuscript in preparation) and F2Ac is a reversible active site directed Shp1 inhibitor (J.M. and Z.Y.Z., manuscript in preparation.

Platelet preparation and functional assays

Blood was collected from the aorta of anesthetized mice into 1/10 acid-citrate-dextrose (ACD) anticoagulant. Platelet counts were measured from peripheral blood samples using an automated hematology analyzer (Element HT5 counter, Heska company). Washed platelets were prepared as previously described.²⁵ Platelet counts were normalized and used for aggregation (2 x 10⁸/ml) or biochemical analysis (5 x 10⁸/ml). Platelet aggregation was measured using the lumi-aggregometer APACT®4004. Surface glycoprotein expression was measured in whole blood by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated antibodies. Resting and activated platelets were fixed and stained with anti-P-selectin antibody.

Megakaryocyte culture and functional assays

Mature MKs from mouse BM were cultured and analysed, as previously described.²⁶ BM cells were obtained from femora, tibiae and iliac crests of C57BL6 mice for pharmacological study and Gp1ba-Cre mice for other experiments by flushing, and Lin-cells obtained after immunomagnetic sorting were cultured for 3 days at 37°C 5% CO2 at a concentration of 1×10⁶ cells/well in a Dulbecco’s Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS), 50 ng/ml Tpo, 100 U/ml Hirudin, 1% PSG.²⁶ MK ploidy was assessed after 3 days of culture, using FITC-coupled anti-CD41 antibody and DNA staining with propidium iodide (PI). Samples were acquired using the BD Fortessa X20 and analyzed using BD FACSDiva software. The ability of MKs to form proplatelets was assessed in vitro, and ex vivo using the explant assay.²⁷

Immunoblotting

Platelet and MK whole cell lysates (WCLs) were prepared and analyzed by automated capillary-based immunoassay (ProteinSimple Jess), prepared according to manufacturer’s instructions, as previously described.²⁸ The High Dynamic Range profile was used for chemiluminescent and fluorescent multiplexing signal detection. Optimized antibody dilutions and sample concentrations used are provided in Supplemental Table 1.

Immunohistochemistry

Spleens from KO and litter-matched CT mice were fixed in buffered formalin and embedded in paraffin. Sections (5 μm) were H&E stained and examined by light microscopy using a 40× objective.

Electron microscopy

Maturation stage of MKs was assessed by electron microscopy (EM). BM samples obtained by flushing mouse femora with 0.1 M sodium cacodylate buffer were fixed in 2.5% glutaraldehyde and embedded in Epon as previously described.²⁹ Thin sections were stained with uranyl acetate and lead citrate, and examined under a JEOL 2100Plus transmission electron microscope at 120 kV (Jeol, Tokyo, Japan). MKs at stages I, II and III were identified using distinct ultrastructural characteristics: stage I, a cell 10–15 μm in diameter with a large nucleus; stage II, a cell 15–30 μm in diameter containing platelet-specific granules; stage III, 40 µm, a MK containing a well-developed demarcation membrane system defining cytoplasmic territories and a peripheral zone. Samples from at least three mice of each genotype were examined in each case.

Platelet recovery assay

An intraperitoneal injection of anti-GPIbα antibody (Emfret) at 2 mg/kg is performed at D0. Blood samples were collected and platelet counts measured prior to injection, and at D5, D10, D15 and D20.³⁰

HSPC colony-forming unit (CFU) assay

Cells are obtained by flushing and were resuspended in IMDM and plated at a density of 1×10⁴ cells per dish in methylcellulose-based medium (MethoCult™ GF M3434, Stemcell Technologies) containing appropriate cytokines to support multilineage hematopoietic colony formation. Cultures were incubated at 37 °C with 5% CO₂ in a humidified incubator for 10–14 days. Colonies were scored under an inverted microscope and classified based on morphological criteria into CFU-G (granulocyte), CFU-M (macrophage), CFU-GM (granulocyte-macrophage), BFU-E (erythroid), and CFU-GEMM (granulocyte, erythrocyte, macrophage, megakaryocyte). Only colonies containing ≥50 cells were counted. Results were expressed as the mean colony number ± SEM from n = 3 independent experiments.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 9 software. Data were expressed as mean ± standard error mean (SEM). Statistical significance was analyzed by one- or two-way ANOVA followed by the appropriate post hoc test, or Kruskal-Wallis test for nonparametric data, a Bonferroni correction was applied for multiple comparisons, as indicated in figure legends. P-values < 0.05 were considered statistically significant.

Results

Deletion of Shp1 and Shp2 in the MK/platelet lineage

To validate the efficiency of protein ablation of Shp1 and Shp2 proteins in MKs/platelets using the Gp1ba-Cre transgenic mouse strain, we employed a quantitative capillary-based immunoassay. This was performed on cells derived from Ptpn6^fl/fl;Gp1ba-Cre^+/KI (Shp1 KO), Ptpn11^fl/fl;Gp1ba-Cre^+/KI (Shp2 KO) and Ptpn6^fl/fl;Ptpn11^fl/fl;Gp1ba-Cre^+/KIdouble-knockout (DKO) mice, with Gp1ba-Cre^+/KI littermates serving as controls.

We firstly evaluated Shp1 and Shp2 levels in MK-sorted progenitors (MKPs) in order to obtain a pure population of matures MKs. Under these conditions, we quantified protein loss of Shp1 and Shp2 in MKs, as shown in Figure 1Ai-iii, with significant 70% and 82% reduction of Shp1 protein in Shp1 KO and Shp1/2 DKO mice, respectively and 78% and 75% reduction of Shp2 protein in Shp2 KO and Shp1/2 DKO mice, respectively compared to controls.

Figure 1.

We next evaluated protein levels in washed platelets and found efficient deletion in Shp1 KO and Shp2 KO platelets, with a significant 90% and 92% reduction of Shp1 protein in Shp1 KO and Shp1/2 DKO mice respectively, and 95% and 96% reduction of Shp2 protein in Shp2 KO and Shp1/2 DKO mice respectively, compared to controls (Figure 1Bi-iii). In addition, there was no upregulation of Shp1 in Shp2-deficient MKs/platelets and vice versa. These results indicate that the Gp1ba-Cre transgene efficiently deleted loxP-flanked Ptpn6 and Ptpn11, resulting in the significant loss of Shp1 and Shp2 in MKs/platelets.

Macrothrombocytopenia and increased bleeding in Shp1/2 DKO mice

Deletion of Shp1 or Shp2 using the Gp1ba-Cre-mouse strain did not affect platelet counts and volumes as shown in Figure 2Ai-ii and Supplemental Table 2. However, Shp1/2 DKO mice were macrothrombocytopenic, with a 40% reduction in platelet count and 15% increase in platelet volume (Figure 2Ai-ii). Monocyte, neutrophil and eosinophil counts were marginally increased in Shp1/2 DKO mice, suggesting possible immune responses. (Supplemental Table 2).

Figure 2.

The primary function of platelets is to prevent blood loss following injury. Therefore, we investigated the effect of the loss of Shp1 and Shp2 on hemostasis. Shp1/2 DKO exhibited a significant increase in bleeding following tail injury as shown in Figure 2Bi-ii. Bleeding time was mildly prolonged compared to control mice, suggesting impaired hemostasis. Quantitative measurement of blood loss revealed a substantial increase in hemoglobin levels (Figure 2Bi-ii).

Aberrant functional responses of Shp1/2-deficient platelets

We next investigated platelet receptor expression by flow cytometry. Receptor expression was normal in Shp1-and Shp2-deficient platelets. However, we found that the collagen activation receptor GPVI and the α2 subunit of the collagen integrin α2β1 were reduced by 25% and 13%, respectively, in Shp1/2-deficient platelets (Figure 2Ci-ii and Supplemental Table 3). All other major surface receptors analyzed were unaltered in these platelets (Supplemental Figure S2 and Supplementary Table 3).

We next checked platelet aggregation by light transmission aggregometry. Shp1- and Shp2-deficient platelets responded normally to all agonists tested (Figure 3A-C). However, consistent with reduced GPVI and α2 surface expression, Shp1/2-deficient platelets exhibited a marginal reduction in aggregation to a low and intermediate concentration of the GPVI-specific agonist CRP (1 and 3 μg/mL), and collagen (3 μg/mL), which signals via GPVI and binds with high affinity to the integrin α2β1 (Figure 3Ai-ii). However, they responded normally to thrombin (0.06 and 0.1 U/ml) (Figure 3Bi-ii), antibody-mediated cross-linking of the hem-ITAM-containing podoplanin receptor CLEC-2 (3 μg/mL) and ADP (3 μM) (Figure 3Ci-ii).

Figure 3.

We also measured P-selectin surface expression in whole blood, as a marker of α-granule secretion. Consistent with reduced GPVI expression, Shp1/2-deficient platelets did not respond to 1, 3 and 10 μg/mL CRP (Figure 3Di), but aggregated normally to 100 and 500 μM PAR-4 peptide (Figure 3Dii).

Aberrant ITAM signaling in Shp1- and Shp2-deficient platelets

Since Shp1 and Shp2 have been implicated in regulating ITAM-containing receptor signaling, we investigated GPVI and CLEC-2 signaling in Shp1- and Shp2-deficient platelets. We focused on SFKs, which phosphorylate tyrosine residues within the ITAM of GPVI-associated FcR γ-chain and hemi-ITAM of the CLEC-2 receptor, and Syk, which docks to both via its tandem SH2 domains, and mediates downstream effects. SFK activation was indirectly measured as trans-autophosphorylation of the highly conserved tyrosine residue 418 in Src (Src p-Tyr418) and Syk activation was measured as trans-autophosphorylation of tyrosine residues 519/520 (p-Tyr519/20), which directly correlate with activity. Western blot analysis demonstrated that Syk p-Tyr519/520 was significantly decreased in CRP–stimulated platelets from Shp1/2-deficient platelets (Figure 4Ai), and marginally reduced in CLEC-2-stimulated Shp1 KO and Shp2 KO deficient platelets. (Figure 4Bi). Notably, Src p-Tyr418 was not altered under any conditions (Figure 4Aii and Bii), suggesting that Shp1 and Shp2 modulate Syk-dependent signaling downstream of GPVI and CLEC-2.

Figure 4.

Impaired megakaryocyte differentiation and function

To determine the cause of thrombocytopenia in Shp1/2 DKO mice, we measured platelet recovery following antibody-mediated depletion as an indicator of platelet turnover. As expected, administration of the anti-GPIbα antibody resulted in extreme and sustained thrombocytopenia, confirming efficient depletion of circulating platelets (Supplemental Figure S3). No differences were observed the different phases or kinetics of platelet clearance or recovery in any of the mouse models. Interestingly, spleen size and weight were significantly increased in Shp1/2 DKO mice (Supplemental Figure S4), suggesting compensatory extramedullary hematopoiesis. However, HSPC colony-forming unit (CFU) assays performed on BM cells showed no significant difference in the frequency or composition of HSPCs, including MKPs between genotypes in vitro (Supplemental Figure S5).

Despite the maintenance of normal platelet turnover in vivo, in vitro analyses revealed pronounced defects in MK development, maturation and function of Shp1/2-deficient MKs (Figure 5A-C). BM-derived MKs from DKO mice exhibited a severe defect in polyploidization, as shown by a significant increase in 2-8N immature MKs, and decreased 16-64N mature MKs (Figure 5Ai-ii). Moreover, proplatelet formation was severely impaired, with a significantly lower percentage of MKs extending cytoplasmic projections compared to controls (Figure 5B).

Figure 5.

Collectively, these data suggest that while homeostatic and compensatory thrombopoiesis is preserved in vivo, potentially supported by extramedullary hematopoiesis, Shp1 and Shp2 are required for efficient megakaryopoiesis in vitro, highlighting a critical role in late-stage megakaryopoiesis under defined conditions.

Reduced Tpo signaling in Shp1/2-deficient MKs

To understand the molecular basis of impaired megakaryopoiesis in Shp1/2 DKO mice, we assessed Tpo-mediated signaling in BM-derived MKs. We previously demonstrated that Shp2 is a positive regulator of ERK1/2 downstream of Mpl. Tpo-mediated ERK1/2 activation was normal in Shp1-deficient MK and significantly reduced in Shp2- and Shp1/2-deficient MKs. However, other downstream effectors, including AKT and STAT3, showed modest or variable changes (Figure 5C). These findings provide a mechanistic explanation for the impaired MK development and maturation observed in vitro, and defective Tpo signaling through the ERK1/2 pathway in Shp1/2-deficient MK, underscoring the importance of these phosphatases in late-stage megakaryopoiesis.

Defective proplatelet formation and MK maturation in Shp1/2-deficient mice

To further characterize the functional consequences of Shp1 and Shp2 deficiency on megakaryopoiesis, we evaluated proplatelet formation ex vivo and examined MK morphology in situ. We first performed ex vivo proplatelet formation assays using BM explants. The results revealed a marked reduction in proplatelet formation in Shp1/2 DKO mice compared with controls, with an increased number of round MKs and a decreased number of MK-forming proplatelets in Shp1/2 DKO mice (Figure 6A-B). To assess MK maturation in situ, we performed EM and ploidy analysis in BM. In control mice, MK displayed characteristic features of terminal maturation, including large polyploid nuclei, demarcation membrane system (DMS) organization, and extensive cytoplasmic development. Shp1/2 DKO MK exhibited a maturation arrest, with increased frequency of stage III MK, defined by incomplete DMS formation, and limited cytoplasmic granularity (Figure 6Ci-ii). This was further supported by flow cytometric analysis of DNA content, showing lower ploidy level (4N–8N) in Shp1/2 DKO MK (Figure 6D). These findings provide further evidence of the involvement of Shp1 and Shp2 in late-stage MK maturation and proplatelet formation, absence of which results in the accumulation of immature, stage III MK in the BM and a functional failure to complete thrombopoiesis.

Figure 6.

Shp2 allosteric inhibitors impair megakaryopoiesis and Tpo signaling

To further delineate the respective roles of Shp1 and Shp2 in MK and Tpo signaling, we employed two pharmacologically distinct inhibitors for each phosphatase to ensure specificity and validate findings from genetically modified mouse models.

For Shp1, we used M029, an covalent allosteric inhibitor that induces conformational changes that block Shp1 activity (Z.Q. and Z.Y.Z., manuscript in preparation), and F2Ac, a reversible inhibitor, which directly target the catalytic site (J.M. and Z.Y.Z., manuscript in preparation). Treatment of murine BM-derived MK with these Shp1 inhibitors showed no significant effects on cell viability, proliferation and MK ploidy (Figure 7Ai-iii), nor on Tpo-mediated signaling (Figure 7B), correlating with what was observed in Shp1-deficient MKs. Similarly, Shp2 was inhibited using SHP099 and RMC-4550, both selective allosteric inhibitors that stabilize Shp2 in its inactive conformation³¹. Addition of Shp2 inhibitors resulted in a marginal decrease in cell viability and a marked reduction of cell proliferation, both established functions of Shp2 in other lineages (Figure 7Ci-ii). Moreover, treatment with either SHP099 or RMC-4550 resulted in a significant inhibition of ploidy with a reduction in the percentage of cells achieving >8N (Figure 7Ciii), and a significant reduction in proplatelet formation (Figure 7Civ). This was accompanied by a severe impairment in Tpo-mediated ERK1/2 and AKT phosphorylation (Figure 7D). This dual-inhibitor profiling reveals that Shp2, but not Shp1, is critical for Tpo-driven MK proliferation, survival, endomitosis and Mpl signaling, highlighting Shp2 as a key regulator of late stage megakaryopoiesis and suggest that its inhibition disrupts thrombopoietic pathways.

Figure 7.

Discussion

In this study, we used the Gp1ba-Cre transgenic mouse model to achieve conditional deletion of the tyrosine phosphatases Shp1 and Shp2 in the MK/platelet lineage. Our results demonstrate efficient deletion of Shp1 and Shp2 in MKs and more completely in platelets, confirming the utility of this mouse model for studying protein involvement in megakaryopoiesis and thrombopoiesis. Residual Shp1 and Shp2 in MKs allowed us to study the phenotype of DKO MKs more thoroughly compared with DKO MKs on the Pf4-Cre background, which were severely compromised developmentally and did not survive in vitro.¹⁵ Additionally, the less severe MK defects meant fewer platelet anomalies, which contained lower levels of Shp1 and Shp2 than the MKs, suggesting different half-lives of the phosphatases in MKs versus platelets. The Gp1ba-Cre transgenic mouse allowed us to reveal distinct and synergistic functions of Shp1 and Shp2 in these processes, without confounding effects of deleting Shp1 and Shp2 in non-MK/platelet lineages observed with the Pf4-Cre transgene mouse.

Deletion of Shp1 or Shp2 alone did not significantly affect platelet counts or volumes, while combined deletion resulted in macrothrombocytopenia characterized by a 40% reduction in platelet counts and marginal increase in platelet volume. This phenotype was accompanied by prolonged bleeding and increased blood loss following tail tip excision, indicating impaired hemostatic function. These defects correlated with reduced GPVI and α2 expression, GPVI-mediated platelet aggregation and P-selectin expression. GPVI signaling was also impaired in Shp1/2-deficient platelets, with reduced Syk phosphorylation despite normal SFK activation, suggesting that Shp1 and Shp2 modulate ITAM/Syk proximal signaling. This is further supported by the marginal reduction in Syk activation downstream of the hemi-ITAM-containing CLEC-2 receptor.

Similar phases and kinetics of platelet recovery following immune-induced platelet clearance suggest that thrombocytopenia in Shp1/2 DKO mice is not due to increased platelet clearance, but rather to impaired MK development, maturation and function. In vitro assays showed significant defects in MK polyploidization and proplatelet formation, while in vivo analysis demonstrated an accumulation of immature MKs with incomplete demarcation membrane system formation and lower ploidy levels. These findings point to a critical requirement for Shp1 and Shp2 in late-stage MK maturation, a process tightly regulated by Tpo signaling through Mpl. Consistently, Tpo-mediated activation of the ERK1/2 pathway was markedly reduced in Shp2- and Shp1/2-deficient MKs, while changes in AKT and STAT3 pathways were modest, underscoring the prominent role of Shp2 in facilitating Tpo-driven Ras/MAPK signaling. Based on these findings, the synergistic effects of deleting both Shp1 and Shp2 suggests that Shp1 acts through another, as yet undefined pathway vital for megakaryopoiesis and thrombopoiesis. RhoA/ROCK is an obvious candidate that Shp1 has been implicated in regulating in other lineages and is essential for cytoskeletal remodeling during endomitosis.

Pharmacological inhibition corroborated genetic findings. Selective inhibition of Shp2 with allosteric inhibitors SHP099 and RMC-4550 significantly impaired MK viability, proliferation, polyploidization, proplatelet formation, and Tpo signaling, while Shp1 inhibitors M029 and F2Ac had no significant effects. This demonstrates a dominant role for Shp2 in regulating MK proliferation and differentiation downstream of Tpo, whereas Shp1 is dispensable for these functions. The pharmacological data thus confirm the functional specificity of the two phosphatases and provide further mechanistic insights into their differential contributions. The milder nature of the platelet and hemostasis phenotypes in Shp1/2 DKO platelets suggests that inhibiting Shp2 will not having any bleeding consequence in patients.

The relatively milder, lineage-specific phenotype of Gp1ba-Cre-driven Shp1/2 DKO mice provides distinct advantages to the previously described Pf4-Cre mouse model ^32,33 (Supplemental Table 4). Pf4-Cre-mediated deletion induces recombination earlier during megakaryopoiesis, starting in MK progenitors, whereas Gp1ba-Cre is activated predominantly in mature MKs and platelets. This temporal difference allows normal early MK development and platelet production, resulting in less severe thrombocytopenia and functional defects. Additionally, our protein quantification data show significant, but incomplete ablation of Shp1 and Shp2 in BM-derived MKs, suggesting residual phosphatase activity is sufficient under steady-state conditions. In contrast, Pf4-Cre models generally achieve near-complete deletion at earlier stages of development, leading to profound thrombocytopenia and often lethality. Furthermore, compensatory extramedullary hematopoiesis observed in our Gp1ba-Cre model may further alleviate thrombocytopenia. Importantly, this milder phenotype allowed us to study the Shp1/2 DKO mice in detail, which was impossible with the Pf4-Cre model due to the severity of the phenotype and cell viability. Findings further establish the utility of the Gp1ba-Cre strain for dissecting the distinct roles of Shp1 and Shp2 in late-stage megakaryopoiesis and platelet biology.

Collectively, Shp2 is a critical positive regulator of Mpl signaling and downstream Ras/MAPK pathways, essential for MK proliferation, polyploidization, and proplatelet formation, whereas Shp1 plays a more modulatory role in ITAM-containing receptor signaling in platelets without directly impacting MK maturation. The combined deletion of Shp1 and Shp2 reveals additive effects on platelet function and MK development, underscoring the importance of coordinated phosphatase regulation in maintaining platelet homeostasis. This study provides the first detailed analysis of Shp1 and Shp2 functions in megakaryopoiesis and platelet biology using a late-stage MK/platelet-specific deletion model, highlighting the indispensable role of Shp2 in megakaryopoiesis and thrombopoiesis, and distinct contributions of Shp1 in platelet signaling. It also emphasizes the utility of the Gp1ba-Cre mouse for exploring protein function in the MK/platelet lineage. These insights may have broader implications for understanding phosphatase regulation in hematopoiesis and could inform therapeutic targeting of Shp2 in hematologic diseases.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgements

This work was supported by Inserm, the Agence Nationale pour la Recherche (AM, ANR-22-REMYS-CE14-0061-01 (E. Barre, PhD candidate), and YS, ANR-23-TARGITT-CE18-0020-01). Z.Q., J. M. and Z.Y.Z are supported by NIH RO1 CA069292. The authors acknowledge all members of the animal facility at UMR_S1255 for maintenance of mouse colonies and the flow cytometry platform (CytoTriCS).

Additional information

Author Contributions

E.B. Performed experiments, analyzed data, revised the manuscript.

MD.LR. Performed experiments, analyzed data, revised the manuscript.

L.Z. Performed experiments and analyzed data.

M.P. Performed experiments and analyzed data.

C.L. Performed experiments and analyzed data.

F.P. Performed experiments and analyzed data.

JY.R. Performed experiments and analyzed data.

A.E. Analyzed data.

Z.Q. Provided reagents.

J.M. Provided reagents.

Z.Y.Z. Provided reagents and revised the manuscript.

Y.A.S. Designed experiments, analyzed data, wrote and revised the manuscript.

A.M. Conceptualized, designed experiments, analyzed data, wrote and revised the manuscript.

Funding

Agence Nationale de la Recherche (ANR) (ANR-22- REMYS-CE14-0061-01)

• Marion Pugliano

• Elsa Barré

• Alexandra Mazharian

Agence Nationale de la Recherche (ANR) (ANR-23-TARGITT-CE18-0020- 01)

• Yotis A Senis

• Marion Pugliano

• Cécile Loubière

HHS | National Institutes of Health (NIH) (RO1 CA069292)

• Zihan Qu

• Zhong-Yin Zhang

• Jinmin Miao

Additional files

Supplemental Figures