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  1. Evaluation Summary:

    Advances in the discovery of novel anti-platelet therapeutics remains an unmet need. This manuscript by Chaurasia et al. describes a novel signaling pathway involving Notch1 and its ligand, Delta-like ligand-4 (DLL4) in driving platelet activation and thrombus formation. The authors provide convincing mechanistic studies to show that blockade of this pathway may serve as a new therapeutic approach to prevent/treat thrombosis. The work will be of great interest to individuals in the hematology and thrombosis field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    The manuscript titled "Notch signaling functions in non-canonical juxtacrine manner in platelets to amplify thrombogenicity" by Chaurasia et al describes that human platelets have notable expression of Notch1 and its ligand DLL-4, which function in a non-canonical manner to synergize with physiological platelet agonists, leading to prothrombotic phenotype. Targeting Notch signaling specifically DLL-4-Notch1-NICD axis can be a potential approach to develop anti-platelet/anti-thrombotic therapeutic.

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  3. Reviewer #2 (Public Review):

    The study by Chaurasia et al addresses an interesting insight to the role of Notch signaling in platelet activation. This is a well-designed and well performed study with some novel findings. Here the authors demonstrate the presence of Notch1 and its ligand DLL-4 in human platelets. Synthesis and surface translocation of these two proteins were found to be upregulated upon stimulation with physiological agonists like thrombin. They show that, DLL-4-mediated platelet activation is associated with cleavage of Notch receptor and generation of its intracellular domain. Further, prothrombotic responses were averted by inhibition of γ-secretase which, too, precluded agonist-induced platelet activation and impaired arterial thrombosis in mice. Platelet aggregation could be inhibited with a blocking antibody that prevented DLL-4-Notch1 interaction. Authors concluded that, non-canonical Notch signaling in platelets propagates in juxtracrine manner within platelet aggregate and synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, targeting Notch signaling can be investigated as a potential anti-platelet/anti-thrombotic therapeutic approach. Author's conclusions were well justified by the data presented in this manuscript.

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  4. Reviewer #3 (Public Review):

    The authors revealed the novel role of the DLL-4-Notch1-NICD signaling axis played in platelet activation, aggregation, and thrombus formation. They firstly confirmed the expression of Notch1 and DLL-4 in human platelets and demonstrated both Notch1 and DLL-4 were upregulated in response to thrombin stimulation. Further, they confirmed the exposure of human platelets with DLL-4 would lead to γ-secretase mediated NICD (a calpain substrate) release. Stimulating platelets with DLL-4 alone triggered platelet activation measured by integrin αIIbβ3 activation, P-selectin translocation, granule release, enhanced platelet-neutrophil and platelet-monocyte interactions, intracellular calcium mobilization, PEVs release, phosphorylation of cytosolic proteins, and PI3K and PKC activation. In addition, Susheel N. Chaurasia et al. showed that when platelets were stimulated with DLL-4 and low-dose thrombin, the Notch1 signaling can operate in a juxtacrine manner to potentiate low dose thrombin mediate platelet activation. When the DLL-4-Notch1-NICD signaling axis was blocked by γ-secretase inhibitors, the platelets responding to stimulation were attenuated, and the arterial thrombosis in mice was impaired.

    This study by Susheel N. Chaurasia et al. was carefully designed and used multiple approaches to test their hypothesis. Their research raised the potential of targeting the DLL-4-Notch1-NICD signaling axis for anti-platelet and anti-thrombotic therapies. Interestingly, compared to thrombin, a potent physiological platelet agonist, the signaling cascade triggered by DLL-4 was relatively weak. Given that the upregulation of DLL-4 and Notch1 happened in response to thrombin stimulation, how much DLL-4 mediated signaling could contribute to in vivo platelet activation in the presence of thrombin is questionable. This could potentially limit the application of targeting Notch1 as an anti-thrombotic therapy. Further, the authors showed that Notch1 signaling could operate in a juxtacrine manner to potentiate low dose thrombin mediate platelet activation, which means the DLL-4 mediated platelet signaling can act as an accelerator of early-stage hemostasis. Again, inhibition of Notch1 may slow down the hemostasis process. But given the fact that there are other platelet agonists (ADP, collagen...) existing simultaneously, blocking Notch1 signaling may not have a strong anti-platelet effect.

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  6. Author Response:

    Reviewer #3 (Public Review):

    The authors revealed the novel role of the DLL-4-Notch1-NICD signaling axis played in platelet activation, aggregation, and thrombus formation. They firstly confirmed the expression of Notch1 and DLL-4 in human platelets and demonstrated both Notch1 and DLL-4 were upregulated in response to thrombin stimulation. Further, they confirmed the exposure of human platelets with DLL-4 would lead to γ-secretase mediated NICD (a calpain substrate) release. Stimulating platelets with DLL-4 alone triggered platelet activation measured by integrin αIIbβ3 activation, P-selectin translocation, granule release, enhanced platelet-neutrophil and platelet-monocyte interactions, intracellular calcium mobilization, PEVs release, phosphorylation of cytosolic proteins, and PI3K and PKC activation. In addition, Susheel N. Chaurasia et al. showed that when platelets were stimulated with DLL-4 and low-dose thrombin, the Notch1 signaling can operate in a juxtacrine manner to potentiate low dose thrombin mediate platelet activation. When the DLL-4-Notch1-NICD signaling axis was blocked by γ-secretase inhibitors, the platelets responding to stimulation were attenuated, and the arterial thrombosis in mice was impaired.

    This study by Susheel N. Chaurasia et al. was carefully designed and used multiple approaches to test their hypothesis. Their research raised the potential of targeting the DLL-4-Notch1-NICD signaling axis for anti-platelet and anti-thrombotic therapies. Interestingly, compared to thrombin, a potent physiological platelet agonist, the signaling cascade triggered by DLL-4 was relatively weak. Given that the upregulation of DLL-4 and Notch1 happened in response to thrombin stimulation, how much DLL-4 mediated signaling could contribute to in vivo platelet activation in the presence of thrombin is questionable. This could potentially limit the application of targeting Notch1 as an anti-thrombotic therapy. Further, the authors showed that Notch1 signaling could operate in a juxtacrine manner to potentiate low dose thrombin mediate platelet activation, which means the DLL-4 mediated platelet signaling can act as an accelerator of early-stage hemostasis. Again, inhibition of Notch1 may slow down the hemostasis process. But given the fact that there are other platelet agonists (ADP, collagen...) existing simultaneously, blocking Notch1 signaling may not have a strong anti-platelet effect.

    We concur with the Public Reviewer that, further study is needed to delineate extent of contribution of DLL-4 signaling in thrombin-activated platelets. However, it is now amply clear that Notch signaling plays a central role in development of thrombinactivated phenotype of platelets. Further, DLL-4-Notch1 interaction on surfaces of adjacent platelets within the thrombus reinforces platelet-platelet aggregate formation. This is further reflected from significant inhibition of thrombus formation in vivo in presence of DAPT in a mouse model of intravital thrombosis. Given that there is a lot of redundancy in stimulation of platelets employing different physiological agonists (ADP, collagen, thrombin etc.), none of the present-day drugs is fully capable of effective platelet inhibition due to parallel signaling pathways. Thus, discovery of Notch signaling and its seminal role in platelet activation could explain redundancy associated with anti-platelet drugs, and Notch inhibition could complement with existing anti-platelet regimen in evoking effective and complete platelet inhibition.

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