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

Reviewer #3 (Public review):

Summary:

In this manuscript, Barré et al utilize the Gp1ba-Cre transgenic mouse model to build upon previous findings in a Pf4-Cre system to investigate the effects of individual and combined Shp1 and Shp2 deletion in megakaryocytes and platelets. They report decreased megakaryocyte maturation, macrothrombocytopenia, and increased blood loss primarily in association with the Shp1/Shp2 double-knockout condition. The authors further show that this phenotype appears to be driven primarily by Shp2 and implicate dysregulation of Tpo signaling and downstream Ras/MAPK pathways, including ERK1/2. They propose that Shp1 may be functioning through a distinct pathway that has yet to be identified, opening up areas for future study.

Strengths:

Overall, the experiments combine in vitro, in vivo, and ex vivo approaches and appear to have been carefully designed and carried out, with multiple technical and biological replicates where relevant. The authors make a compelling argument for using the Gp1ba-Cre as opposed to the Pf4-Cre system and demonstrate both the dose- and stage-dependent effects of Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis. They find that Shp1 and Shp2 are required in late-stage megakaryocyte maturation and that even low levels of expression compared to baseline are likely sufficient to yield generally normal megakaryocytes. Their findings also lead to specific future directions, such as the mechanism by which Shp1 regulates megakaryopoiesis and thrombopoiesis that is distinct from Tpo-mediated signaling. Figure 8 is particularly effective in summarizing the different models and pathways presented.

Weaknesses:

The effects of Shp1 and Shp2 knockouts are described as "synergistic," but it is not always clear that the effects are synergistic vs. additive, especially as the specific mechanism by which Shp1 functions in megakaryocyte development has yet to be identified. On a more minor point, although a significant part of the introduction focuses on the role of Mpl signaling in human disease, there is ultimately limited reference to Mpl (although there is of course a strong focus on Tpo) and the potential clinical implications of the findings presented here.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment

This manuscript provides an important contribution to the field of platelet biogenesis, and the convincing evidence will advance our understanding of signal transduction driving the development of late megakaryopoiesis and platelet reactivity that results in bleeding diathesis. The paper is noteworthy for analyzing two related, either singly or in combination, tyrosine phosphatases in this conditional, stage development gene knockout. Because SHP1 is a negative regulator and SHP2 is an activator, the synergistic effects found in the double knockout were surprising.

We thank the reviewer for acknowledging the importance and novelty of our findings.

Public Reviews:

Reviewer #1 (Public review):

Barré et al. investigated the role of Shp1 and Shp2 in megakaryocytes (MKs) and platelets by conditional knock-out of Shp1, Shp2, or both under the control of the Gp1ba promoter. Deletion of Shp1 and Shp2 in MKs and platelets was almost complete. The Shp1/Shp2 double knock-out mice displayed macrothrombocytopenia and increased bleeding, whereas the single knock-outs did not show significant defects. Platelet function was aberrant in DKOs, but not in single knock-outs, and so was ligand-induced signaling, particularly Syk phosphorylation.

Megakaryocyte maturation was impaired in Shp1/Shp2 DKO mice. Ligand-induced signaling was impaired in Shp2 knock-out and DKO. Ex vivo formation of platelets and in vivo maturation of MKs were impaired in DKO mice. Pharmacological inhibitors of Shp1 and Shp2 had largely similar effects as observed in the single knock-outs. The authors conclude that Shp1 and Shp2 have synergistic functions in the MK/platelet lineage, and that Shp2 may be a potential therapeutic target in myeloproliferative neoplasms.

Strengths:

The data clearly show effects of the Shp1/Shp2 double knock-out on MKs and platelets.

Weaknesses:

There appears to be a discrepancy between the results with the Shp2 single knock-out and the Shp2 inhibitor: the Shp2 knock-out does not affect MKs and platelets, except Erk1/2 signaling, whereas the Shp2 inhibitors appear to affect MK function.

This work is interesting and may have potential from a therapeutic point of view.

Pharmacological effects do not always correlate with congenital anomalies arising for genetic defects. The Shp2 allosteric inhibitors used in our study only inhibit catalytically inactive Shp2, whereas targeted deletion of Ptpn11 results in a loss of total Shp2 expression, including catalytic and non-catalytic related functions, with developmental consequences. Further, Gp1ba-Cre+; Shp2fl/fl megakaryocytes express approximately 22% of normal Shp2 level, which likely also contributes to differences observed between pharmacological inhibition and genetic ablation of Shp2.

We thank the reviewer for recognizing the therapeutic potential of our findings.

Reviewer #2 (Public review):

Summary:

In this manuscript, Barré et al. investigate the roles of the phosphatases Shp1 and Shp2 in the megakaryocyte and platelet lineage using genetic depletion in mice. By employing Gp1ba-Cre-based models, the study builds on the authors' previous work and addresses some limitations associated with earlier Pf4-Cre approaches. The authors report relatively mild alterations in megakaryocyte and platelet parameters in mice lacking either Shp1 or Shp2 alone, whereas combined deletion of both phosphatases results in macrothrombocytopenia, mild bleeding, and impaired GPVI-dependent platelet aggregation accompanied by reduced Syk phosphorylation. The functional platelet defects are linked to reduced expression of GPVI and integrin α2, while thrombocytopenia is associated with impaired megakaryocyte maturation, reduced ploidy, defective proplatelet formation, and altered TPO-dependent Ras/MAPK signaling. Similar effects on megakaryopoiesis are also observed in vitro following treatment with newly developed Shp2 inhibitors.

Strengths and Weaknesses:

The study addresses an important biological question and presents a substantial dataset that could contribute to a better understanding of Shp1 and Shp2 function in platelet biology. However, several aspects of data presentation and interpretation would benefit from additional clarification. In particular, while the authors conclude that single genetic deletion or pharmacological inhibition of Shp1 has a limited impact and that the major phenotypes are specific to combined Shp1/2 deletion or Shp2 inhibition, some of the data suggest more nuanced effects that may warrant further discussion.

We thank the reviewer for raising this point. The manuscript is being revised accordingly, including highlighting the potential role of Shp1 in megakaryopoiesis and thrombopoiesis under steady-state and stressed conditions, requiring more detailed investigation.

Reviewer #3 (Public review):

Summary:

In this manuscript, Barré et al utilize the Gp1ba-Cre transgenic mouse model to build upon previous findings in a Pf4-Cre system to investigate the effects of individual and combined Shp1 and Shp2 deletion in megakaryocytes and platelets. They report decreased megakaryocyte maturation, macrothrombocytopenia, and increased bleeding primarily in association with the Shp1/Shp2 double-knockout condition. The authors further show that this phenotype appears to be driven primarily by Shp2 and implicate dysregulation of Mpl signaling and downstream Ras/MAPK pathways, including ERK1/2. Given the key role of these pathways in human diseases such as myeloproliferative neoplasms and the challenges associated with modulating such a central pathway, identification of a specific regulator of Mpl signaling poses intriguing questions for future studies on clinical applicability.

We thank the reviewer for acknowledging the importance and novelty of our findings.

Strengths:

Overall, the experiments combine in vitro, in vivo, and ex vivo approaches and appear to have been carefully designed and carried out, with multiple technical and biological replicates where relevant. The authors make a compelling argument for using the Gp1baCre as opposed to the Pf4-Cre system and demonstrate both the dose- and stagedependent effects of Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis. They find that Shp1 and Shp2 are required in late-stage megakaryocyte maturation and that even low levels of expression compared to baseline are likely sufficient to yield generally normal megakaryocytes. Their findings also lead to specific future directions, such as the mechanism by which Shp1 regulates megakaryopoiesis and thrombopoiesis that is distinct from TPO-mediated signaling.

Weaknesses:

While the experiments have been thoughtfully designed and carried out, there is limited background explanation on relatively complex or niche pathways/mechanisms, such as the relationship between P-selectin, CRP, and PAR4p; the interactions between SFK, Syk, GPVI, and CLEC-2; and TPO, MPL, ERK1/2, AKT, and STAT3, which, while likely intuitive to experts in their respective fields, may be less obvious to a reader approaching this manuscript with a global interest in megakaryopoiesis/thrombopoiesis and thus detract from the impact of the findings.

We thank the reviewer for raising this point. The manuscript is being revised to better explain the rationale and molecular mechanisms linking these pathways and functions.

With regard to the science itself, some of the conclusions feel premature based on the available data.

(1) The section "Aberrant ITAM signaling in Shp1- and Shp2-deficient platelets" is challenging to follow for those not well-versed in ITAM signaling and associated pathways, and may take additional outside reading to follow the conclusion that Syk-dependent signaling is modulated downstream of GPVI and CLEC-2 based on lack of change in Src p-Tyr418, especially considering that Src p-Tyr418 was previously introduced as a measure of SFK rather than Syk. In the introduction, Shp1 is specifically mentioned as a negative regulator of the ITAM/Syk/phospholipase pathway. However, in Figure 4Ai and Bi, Syk phosphorylation/activation in Shp1 knockout cells did not appear to be different from Shp2 knockout cells, and is lower than the control, which is surprising for a negative regulator. It is also not clear why, in the section (Figure 4A-B), there is reduced Syk activation in Shp1 and Shp2 single knockout cells upon CLEC2 stimulation (but apparently not with CRP) when there was no difference in response to CLEC2 (but a difference in response to CRP) in the previous section (Figure 3A, C).

We thank the reviewer for raising these important points. The manuscript is being revised accordingly, including clarifying the roles of SFKs, Shp1 and Shp2 in the ITAM-Syk-PLCγ2 signaling pathway.

Briefly, SFKs are essential for phosphorylating ITAMs, allowing SH2-dependent docking of Syk. Reduced reactivity of Shp1/2 DKO platelets to CRP and collagen is likely due to downregulation of the ITAM-containing GPVI-FcR γ-chain complex and integrin α2 subunit, and concomitant reduction in Syk phosphorylation.

However, the marginal albeit significant reduction in Syk phosphorylation downstream of CLEC-2 in Shp1 and Shp2 KO platelets was not determined and was insufficient to impact CLEC-2-mediated platelet aggregation under the conditions tested.

Differences in the stoichiometry and docking of Syk to phosphorylated GPVI-FcR γ-chain and CLEC-2 likely contribute to the differences in platelet reactivity and Syk phosphorylation downstream of the two receptors in the absence of Shp1 and Shp2.

(2) In the section "Reduced Tpo signaling in Shp1/2-deficient MKs," only Western blot data for (p)ERK1/2, AKT, and STAT3 are presented before concluding that decreased ERK1/2 activity is a mechanistic explanation for thrombocytopenia seen in the Shp1/2 doubleknockout condition. Such a statement would benefit from additional experiments, such as protein or transcriptional levels of ERK1/2 targets specifically relevant to megakaryopoiesis, such as ETS, FOS, and JUN, to assess the consequences of decreased phosphorylated ERK1/2.

We thank the reviewers for these constructive comments. Further experiments are being planned to determine the biological and transcriptional consequences of reduced ERK1/2 phosphorylation during megakaryopoiesis and thrombopoiesis.

(3) Suggesting that "inhibiting Shp2 will not have any bleeding consequence in patients" and that Shp2 may be a therapeutic target in myeloproliferative neoplasms when none of these studies have been carried out in a human model is a bold conclusion. There are no data presented on, for example, whether Shp2 inhibition can help reverse the MPL/JAK/STAT pathway in the setting of gain-of-function mutations specifically associated with myeloproliferative neoplasms.

This conclusion is being tempered in the revised manuscript. Genetic- and pharmacological-based approaches will be used to establish the therapeutic potential of inhibiting Shp1 and Shp2 in mouse models of MPN, including Jak2 gain-of-function mice. Bleeding and thrombotic complications of inhibiting Shp1 and Shp2 will be explored as part of these studies.

Recommendations for the authors:

Reviewing Editor Comments:

Altogether, we feel that this is an important study for those in the fields of hematology or signal transduction. Your important study characterizes the roles in late megakaryopoiesis and platelet biogenesis of single or combined conditional deletion of two tyrosine phosphatases, Shp1 and Shp2. Strengths include technical advances in single and combined deletions, the somewhat surprising results of synergy between the two phosphatases, focusing on the critical stage of late megakaryopoiesis, and clinical implications in bleeding diathesis.

Weaknesses are mostly minor, but the numerous points raised by reviewer 3 need to be addressed and typographical errors corrected. Further discussion should include the relevance or dissimilarity in megakaryopoiesis and platelet biogenesis between murine and human blood health and disease. Since SHP1 is a negative regulator and SHP2 is a positive activator, additional discussion about how they coordinate and fine-tune ("nuanced") signal transduction in TPO- or GPVI-induced signaling in an explicitly stated pathway.

We invite you to respond to the critiques and submit a revised manuscript.

Sincerely,

Seth Corey, MD MPH

We thank the editor for the positive evaluation of our study and for highlighting its relevance to the fields of haematology and signal transduction. We have carefully addressed all comments raised by Reviewer 3 and corrected typographical errors throughout the manuscript.

As suggested, we expanded the Discussion to better address the relevance of our murine findings to human megakaryopoiesis and platelet biogenesis. While our study relies on mouse models, key components of TPO/MPL signaling and platelet production are conserved between mice and humans, although differences in megakaryocyte maturation dynamics and platelet biology are acknowledged and now discussed.

We also clarified the coordinated roles of Shp1 and Shp2 in signaling. Although Shp1 generally acts as a negative regulator and Shp2 as a positive mediator of signal transduction, our results suggest that they function in a complementary manner to optimize signaling downstream of TPO/MPL and GPVI pathways, thereby ensuring appropriate regulation of late megakaryopoiesis, platelet production and activation.

These additional considerations have been incorporated into the revised manuscript to provide a clearer conceptual framework for how Shp1 and Shp2 cooperate to regulate platelet biogenesis.

Reviewer #1 (Recommendations for the authors):

(1) The effects of the Shp1/Shp2 DKO are clear, but the effect of the Shp2 single knock-out is less clear on all parameters that were tested. The exception is ERK1/2 phosphorylation, which was reduced in the Shp2 knock-out as well as the Shp1/Shp2 DKO. Why do the authors conclude that Shp2 may be a potential therapeutic target, while the data show that knock-out of Shp1 and Shp2 is required for the observed effects?

We agree that the most pronounced phenotypes were observed in the Shp1/Shp2 DKO. However, Shp2 single knock-out consistently reduced ERK1/2 phosphorylation, indicating that Shp2 contributes to MPL downstream signaling in megakaryocytes. The absence of a strong phenotype in Shp2 single knock-out may be due to residual Shp2 protein. However, given the established role of the Shp2–ERK pathway in megakaryopoiesis and the observation that pharmacological Shp2 inhibition significantly affected MK ploidy, proplatelet formation, and ERK1/2 phosphorylation, our data support a contribution of Shp2 to these processes and suggest it as a potential therapeutic target.

(2) Inhibitors of Shp1 and Shp2 had largely similar effects as Shp1 and Shp2 single knock-outs, respectively. The effect of Shp2 knock-out on MK ploidy is not clear, cf. Figure 5Ai (no effect) and Figure 5Aii (reduction, which is not significant), whereas a clear and significant effect was reported for the Shp1/Shp2 DKO. In contrast, in Figure 7Ciii, the Shp2 inhibitors SHP099 and RMC-4550 clearly affect MK ploidy and the percentage of MKs forming proplatelets. The discrepancy between the effect of Shp2 knock-out and Shp2 inhibitors suggests that the inhibitors may affect other targets. The authors should consider using the Shp2 inhibitors on the Shp2 knock-out to prove or disprove that the effects of the Shp2 inhibitors are mediated exclusively by Shp2.

Pharmacological inhibition does not necessarily phenocopy genetic deletion. The allosteric Shp2 inhibitors used in our study (SHP099 and RMC-4550) stabilize Shp2 in an inactive conformation and inhibit its catalytic activity, whereas Ptpn11 deletion results in complete loss of the Shp2 protein, including both catalytic and scaffolding functions. These mechanistic differences may lead to distinct biological outcomes and could explain the discrepancy observed between Shp2 knockout and inhibitor treatments.

(3) Since the most profound effects were found in the Shp1/Shp2 DKO, it would be interesting to use combinations of the Shp1 and Shp2 pharmacological inhibitors to mimic the effect of the Shp1/Shp2 DKO.

We thank the reviewers for these constructive comments. Further experiments are indeed being planned to use combinations of the Shp1 and Shp2 pharmacological inhibitors to mimic the effect of the Shp1/2 DKO.

Reviewer #2 (Recommendations for the authors):

Major points:

(1) Additional details on the strategy used to isolate megakaryocyte progenitors from mouse bone marrow would improve clarity, including sorting approach, gating strategy, and assessment of population purity.

We thank the reviewer for this suggestion. We have now expanded the Methods section to provide a more detailed description of the strategy used to isolate megakaryocyte progenitors from mouse bone marrow.

Briefly, bone marrow cells were first enriched for hematopoietic progenitors and stained with antibodies against lineage markers and megakaryocyte-associated markers. Megakaryocyte progenitors were then isolated by flow cytometric sorting based on established surface marker combinations, including c-Kit and CD41 expression. The gating strategy excluded lineage-positive cells and debris before selecting the progenitor population of interest.

(2) Platelet GPVI expression appears reduced not only in Shp1/2 double-knockout mice but also, to some extent, in single Shp1- or Shp2-deficient models. A more detailed quantitative comparison and discussion would be helpful.

We thank the reviewer for this observation. Although the most pronounced reduction in GPVI surface expression was observed in Shp1/Shp2 double knock-out platelets, minor variations may appear in the single knock-out models. To address this, we performed additional statistical analyses comparing WT platelets with each single knock-out genotype. These analyses did not reveal any significant statistical differences in GPVI expression between WT and either Shp1- or Shp2-deficient platelets, indicating that the apparent variations fall within the range of biological variability.

(3) The aggregation traces shown in Figures 3A and 3B would benefit from clarification regarding their representativeness relative to the corresponding quantitative analyses.

We thank the reviewer for this comment. The aggregation traces in Figures 3A and 3B represent experiments selected from independent replicates included in the quantitative analysis. The figure legends have been revised to clarify that these traces are representative of the experiments summarized in the quantification panels, which include data from multiple independent mice.

(4) In several experiments, statistical significance may be influenced by differences in sample size across genotypes (e.g., Figures 2Ci, 3Ai, 3Di, and 6Ai). Using comparable numbers of replicates would strengthen the interpretation.

We appreciate the reviewer’s attention to statistical rigour. The differences in sample size between genotypes reflect the availability of animals from the different breeding cohorts. Importantly, all statistical analyses were performed using appropriate tests that account for unequal sample sizes. The observed differences remain consistent across independent experiments.

(5) The rationale for assessing only P-selectin exposure following CRP and PAR4p stimulation is not fully explained. Including integrin αIIbβ3 activation, or clarifying its exclusion, would provide a more complete assessment of platelet activation.

We thank the reviewer for this suggestion. P-selectin exposure was used as a primary readout because it provides a robust measure of α-granule secretion downstream of GPVI and PAR signaling. Integrin αIIbβ3 activation was not assessed in these experiments because platelet aggregation assays were performed in parallel, which already provide a functional readout of integrin activation, as aggregation requires αIIbβ3 engagement. Nonetheless, we agree with the reviewer that direct measurement of integrin activation (e.g., fibrinogen binding) would provide complementary information and will be considered in future studies.

(6) Figure 3Dii is described as an aggregation assay, although it appears to report P-selectin exposure; this distinction should be clarified.

We thank the reviewer for identifying this inconsistency. Figure 3Dii reports indeed P-selectin exposure measured by flow cytometry, rather than platelet aggregation. We have corrected the description in the Results section.

(7) The suggestion of compensatory extramedullary hematopoiesis based on splenomegaly would be strengthened by immunophenotypic analysis of splenic hematopoietic progenitor populations.

We appreciate this important suggestion. In the current study, the evidence for possible compensatory extramedullary hematopoiesis is mainly based on the splenomegaly observed in Shp1/2 DKO mice. We agree that detailed immunophenotypic analysis of splenic hematopoietic progenitors would provide additional mechanistic insight; however, this was beyond the scope of the present study, which focuses on the intrinsic role of Shp1 and Shp2 in the megakaryocyte and platelet lineage. We have therefore revised the Discussion to present this interpretation more cautiously and to indicate that further studies will be required to determine whether splenic hematopoiesis contributes to compensatory platelet production in this model.

(8) In Figure S3, differences in platelet recovery kinetics among genotypes appear evident. Clarification of the statistical tests used to assess these differences would be useful.

We thank the reviewer for this comment. Platelet recovery kinetics were analyzed using two-way ANOVA with appropriate post hoc tests. No statistically significant differences between genotypes were observed. These details have been added to the Methods and figure legend for clarity.

Reviewer #3 (Recommendations for the authors):

Overall, the manuscript suffers from multiple typographical and grammatical errors that distract from the data being presented.

We have carefully revised the manuscript to correct typographical and grammatical errors throughout, improving clarity and readability.

(1) Figure S1: I believe this should be referenced in the first paragraph of the results section.

We have now referenced the Supplemental Figure S1 in the first paragraph of the results section as suggested.

(2) Figure 2A: Although the individual points for the replicates are informative, they do make it difficult to appreciate the SEM, and to my eye it appears that, for example, there may not be a difference between Shp2 and Shp1/2 or that there may be a difference between Shp1 and Shp1/2 in (ii), as Table S2 suggests. In other words, it seems that the increased MPV (as well as the leukocyte phenotype) may be driven by the knockout of Shp2; are there statistical analyses that could be performed to show that the increased MPV is specific to the double knockout?

We thank the reviewer for this comment. Despite the slightly higher MPV observed in Shp2 single knockouts, statistical analysis using one-way ANOVA, which is appropriate for comparing means across multiple independent groups, and taking all individual data points into account, revealed no significant differences between Shp2 or Shp1 single KO and the Shp1/2 DKO.

(3) Figure 2Bi: Is this missing a statistical significance bar, or was there no significant difference in cumulative bleeding time between the conditions? If the latter, this should be clarified in the main text (although the specific sentence regarding bleeding time only claims "mildly prolonged," the preceding sentence indicates "significant increase in bleeding").

Thank you for this comment. There was no statistically significant difference in cumulative bleeding time between the groups. We have now modified the text accordingly to clarify this point and to indicate that, while bleeding time was not significantly different, blood loss was significantly increased in Shp1/2 DKO mice.

(4) Figure 2Ci: What was the extent (statistically) of GPVI reduction in the Shp1 and Shp2 single knockout mice compared to the control? It seems that although there was no change in alpha2 expression in the single-knockout conditions, the contributions of Shp1 and Shp2 loss may be additive on GPVI (although I acknowledge that this is not necessarily borne out in Figure 3Ai).

Thank you for this comment. After reanalyzing the data using an appropriate statistical test (one-way ANOVA followed by Tukey’s post hoc test), we found that GPVI expression is significantly reduced in both Shp1 and Shp2 single knockout platelets compared with controls. However, this reduction did not result in detectable functional consequences on platelet aggregation, as shown in Figure 3Ai.

(5) Figure 3Ai: It seems that the individual replicates for the Shp1/2 double knockout cluster in two populations, extreme non-responders and arguably normal responders to CRP. Are there any biological or technical explanations for this?

We thank the reviewer for this observation. We agree that the distribution of individual replicates in the Shp1/2 DKO group suggests the presence of two subpopulations, with some samples showing markedly impaired aggregation and others retaining near-normal responsiveness to CRP. While all experiments were performed under standardized conditions, subtle differences in platelet preparation, agonist sensitivity, or assay timing could also contribute to dispersion within this group. Importantly, despite this variability, the overall trend indicates a significant reduction in aggregation in the Shp1/2 DKO condition compared to controls, supporting a critical and partially redundant role for Shp1 and Shp2 in GPVI-mediated platelet activation.

(6) "Aberrant functional responses of Shp1/2-deficient platelets": It may be helpful, in the last paragraph of this section, to briefly explain the relationship between P-selectin, CRP, and PAR4p. If short on space/words, the introduction likely does not need an explanation of platelet function and definitions of megakaryopoiesis and thrombopoiesis.

We thank the reviewer for this suggestion. We have revised the last paragraph to clarify that P-selectin surface expression reflects α-granule secretion following platelet activation. We now specify that CRP activates platelets via GPVI signaling, whereas PAR-4 peptide signals through thrombin receptors, providing context for the differential responses observed in Shp1/2-deficient platelets.

(7) "Aberrant ITAM signaling in Shp1- and Shp2-deficient platelets": Is there a cartoon figure panel that could be added to clarify how SFK (which, as an aside, is not defined as an acronym), Syk, GPVI, CLEC-2 receptor, Shp1, and Shp2 are interrelated? In addition to the comments left in the public review, I was perplexed by Figure 4Bi, as the band for the Shp1/2 double knockout condition appears to be stronger than the other 3 conditions, but this is not what is depicted in the bar graph on the right.

We thank the reviewer for this helpful comment. We have now added a schematic cartoon (new Figure 8) to clarify the relationships between SFKs, Syk, GPVI, and the regulatory roles of Shp1 and Shp2. All acronyms, including SFK, are now defined at first mention to improve accessibility.

Regarding Figure 4Bi, we appreciate this observation. The apparent discrepancy between the representative blot and the quantification reflects variability across experiments. The bar graph represents the average of independent replicates.

(8) I would also recommend considering reshuffling the panels in Figure 4 so that the 2 assays measuring Syk phosphorylation and the 2 assays measuring Src phosphorylation are next to each other, as opposed to grouped by agonist. They should also be presented in the order of the text, which states that SFK activation was measured via Src before mentioning Syk (but the data are presented in reverse).

We thank the reviewer for this suggestion. We have reorganized Figure 4 so that the panels measuring Src and Syk phosphorylation are presented together and, in the order, described in the text. The manuscript text has also been updated accordingly to match the revised figure layout.

(9) GPVI overexpression experiments in these megakaryocytes or, conversely, Syk inhibition in control cells, to reverse or recapitulate the phenotype, respectively, may be additionally informative.

We thank the reviewer for this suggestion. We agree that modulating GPVI or Syk activity could provide additional mechanistic insight. While these experiments were beyond the scope of the current study, we plan to explore GPVI overexpression and Syk inhibition in follow-up studies to further validate the pathway’s role in the observed phenotype.

(10) "Reduced Tpo signaling in Shp1/2-deficient MKs": In addition to the comments left in the public review, I would suggest moving this section to after "Defective proplatelet formation and MK maturation in Shp1/2-deficient mice" so that the 2 sets of proplatelet and ploidy data are consecutively presented.

We thank the reviewer for this helpful suggestion. We have now revised the manuscript accordingly by reorganizing both the text and figures. The ploidy and proplatelet formation data are now presented together in Figure 5, followed by the Tpo signaling data in Figure 6, improving the overall flow and clarity of the results section.

(11) Figure 6Cii: Why does Shp1 add up to >100%?

The reason the Shp1 bar exceeds 100% is due to how the data were quantified and normalized. Each segment represents the mean from separate experiments. Stacking these means can exceed 100% because the sum of averages is not equal to the average of the total.

(12) Figure 7D: How do you reconcile these findings of impaired AKT phosphorylation with the addition of a Shp2 inhibitor but no change with Shp2 knockout (Figure 5C)? Would you attribute it to the residual Shp1 and Shp2 in the Cre-Lox MKs?

Pharmacological effects do not always correlate with congenital anomalies arising for genetic defects. The Shp2 allosteric inhibitors used in our study only inhibit catalytically inactive Shp2, whereas targeted deletion of Ptpn11 results in a loss of total Shp2 expression, including catalytic and non-catalytic related functions, with developmental consequences. Further, Gp1ba-Cre+; Shp2fl/fl megakaryocytes express approximately 22% of normal Shp2 level, which likely also contributes to differences observed between pharmacological inhibition and genetic ablation of Shp2.