Peer Review History

Original SubmissionJuly 30, 2020
Decision Letter - Maya Capelson, Editor

Dear Dr Comer,

Thank you for submitting your manuscript entitled "COVID-19 induces a hyperactive phenotype in circulating platelets" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff [as well as by an academic editor with relevant expertise] and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by August 13th, 2020.

Login to Editorial Manager here: https://www.editorialmanager.com/pbiology

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Given the disruptions resulting from the ongoing COVID-19 pandemic, please expect delays in the editorial process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Maya Capelson,

PLOS Biology

Revision 1
Decision Letter - Paula Jauregui, PhD, Editor

Dear Dr. Comer,

Thank you very much for submitting your manuscript "COVID-19 induces a hyperactive phenotype in circulating platelets" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers. They all think that you need to improve some aspects before publication.

In particular, both referees and the Academic Editor want to know if the COVID-19 patients were on aspirin. Referee #1 asks about the MPV and platelet activity status in patients with thrombosis and the comparison to those who did not develop clots, considers that the component of the hyperactive platelets needs to be demonstrated (platelet aggregation assays and thrombotic assays), and asks for other factors that were released by the hyperactive platelets and whether a particular factor correlates with disease prognosis. Referee #1 also considers that the authors need to measure P-selectin on the platelet surface instead of soluble, that they need to explain why TPO levels are elevated but there is no thrombocytosis or increased platelets in patients, and proper controls with patients with equal inflammatory states need to be performed. Finally, referee #1 thinks that you need to directly link the hyperactivity of platelets to the hypercoagulable state in COVID-19. Referee #2 asks whether the in-patient control group had a history of thrombotic events, inflammatory diseases/infections, considers that the authors need to comment on fibrinogen levels, and that they need to add details to the methods. In addition, the Academic Editor has kindly suggested to add a flowchart explaining the flow between figures 1, 2, and 3 to help understand why the measures have been performed in a selection of the patients.

In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

We expect to receive your revised manuscript within 3 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Paula

---

Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

*****************************************************

REVIEWS:

Reviewer's Responses to Questions

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #1: In this manuscript by Comer and colleagues, they present very timely work on the hyperactivity of platelets in COVID-19. This is of interest since there is a high degree of thromboembolic disease in COVID-19 and even with anticoagulation, patients still at risk of thrombosis. In this manuscript the authors demonstrate that patients with COVID-19 have hyperactive circulating platelets and they suggest that this may contribute to the hypercoabulability seen in these patients.

A few concerns that I have include:

1. A large proportion of the patients with more severe presentations of COVID-19 often present with cardiovascular disease such as hypertension or CAD. Since these patients are usually placed on aspirin at baseline and since this manuscript is suggesting the platelet activity is linked to disease severity; it begs the question of whether or not any of these patients were on aspirin. Where there patients who developed covid who were also taking aspirin? Did those patients have hyperactive platelets? Did they have increased thrombotic events?

2. The authors provide information on the mean platelet volume and platelet reactivity in COVID-19 patients. Further analysis of MPV and platelet reactivity as it directly relates to development of a thromboembolic event would directly link hyperactive platelets and hypercoagulability. What was the MPV and platelet activity status in patients with thrombosis and was it different in comparison to those who did not develop clots.

3. Although the platelets were hyperactive by the authors description, a functionality component needs to be demonstrated. Were there changes in platelet aggregation assays? What about thrombotic assays?

4. The authors discuss agonist-induced ADP release. Platelets carry a myriad of inflammatory chemokines and also coagulation factors. What other factors were released from these hyperactive platelets and does a particular factor correlate with disease prognosis?

5. The authors measure soluble P-selectin. P-selectin can come from activated platelets but also activated endothelial cells. We know that COVID-19 is associated with enhanced endothelial injury. The authors should directly measure P-selectin on the platelet surface instead of just soluble P-selectin as a more direct measure of platelet hyperactivity.

6. The authors point out that there is no thrombocytosis in patients. Yet, the TPO levels and MPV are increased which would suggest that the bone marrow is revved up for making platelets. What is keeping the platelet count from becoming elevated? We know that platelets are increased in inflammatory conditions, why is it not increased in COVID which is associated with high levels of inflammation? This especially needs to be explained since the TPO levels are elevated in their study.

7. Although there were differences in the hypereactivity of patients in the nonsevere and severe groups, proper controls with patients with equal inflammatory states need to be performed. Do patients with other highly inflammatory conditions also have platelet hypereactivity? Is this a specific phenomemon on COVID-19.

8. The authors need to directly link the hyperactivity of platelets to the hypercoagulable state in COVID-19

Reviewer #2: This is a well written paper that is highly relevant to the current COVID-19 pandemic. The authors demonstrate differences in routine blood parameters that were associated with disease severity. Platelets form patients with COVID-19 were more prone to activation, demonstrated by ADP release and soluble markers of activation. This study is limited by a small sample size as discuss within the manuscript, nonetheless these are interesting observations that could prove to be clinically useful during the current pandemic.

I have some points to address as detailed below.

What are the in-patient control group, did any of the patients have a history of thrombotic events, have inflammatory diseases/infections?

The healthy controls were not on aspirin/anti-platelet agents - what about the in-patient group?

Unless I missed it, no reference is made in the text is made to supplementary Table 1

The methods require some more details and some clarification.

* Please state number of patients recruited per group

* ACD Vacutainer solution A/B?

* Presumably the pH was adjusted with a buffer, eg. modified tyrodes or hepes etc during washing. The methods state it was with ACD?

* What buffer were the isolated platelets resuspended in? In the platelet ATP secretion assay platelet washing is described? If this was not different to the above this information only needs to be stated once

Are data mean +/- IQR? Please state in figure legends.

Can the authors comment on the fibrinogen levels - in other studies these are reportedly raised on admission.

Typo page 14 - appear to be associated idwith significant morbidity

Comments from the Academic Editor:

It is not clear enough why what measurement was done in which patient. There needs to be more information available on this, and that information is relevant to understanding where the data come from. I am not saying that there need to be more measurement done, but it has to be justified why the measures have been performed in a selection of the patients. A simple flowchart explaining the flow between fig 1, 2, and 3 would already help a lot.

Revision 2

Attachments
Attachment
Submitted filename: Responses to Reviewer Comments.docx
Decision Letter - Paula Jauregui, PhD, Editor

Dear Dr. Comer,

Thank you for submitting your revised Research Article entitled "COVID-19 induces a hyperactive phenotype in circulating platelets" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor. 

Based on the reviews, we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. Please also make sure to address the data and other policy-related requests noted at the end of this email.

The Academic Editor thinks that the explicit reasons for the selection of patients for the following analyses should be stated explicitly. This is not clear in the Supplementary figure 1 and we suggest that you change this flowchart for a CONSORT style flowchart to give full insight into the selection. Please also complete all the requirements that you will find below my signature in this letter.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

-  a cover letter that should detail your responses to any editorial requests, if applicable

-  a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

-  a track-changes file indicating any changes that you have made to the manuscript. 

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information  

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods 

Please do not hesitate to contact me should you have any questions.

Sincerely,

Paula

---

Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

---------------------

DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797 

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. 

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figures 1A, 1B, 2, 3A, 3B, 3C and Supplementary figures 2A and B.

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

 ---------------------------

Reviewer remarks:

Reviewer #1: The authors have answered all of my queries and have appropriately revised the manuscript to address concerns raisedd

Reviewer #2: The authors have adequately addressed all my concerns and significantly improve the manuscript.

Revision 3

Attachments
Attachment
Submitted filename: Responses to Reviewer Comments.docx
Decision Letter - Paula Jauregui, PhD, Editor

Dear Dr. Comer,

On behalf of my colleagues and the Academic Editor, Bob Siegerink, I am pleased to say that we can in principle offer to publish your Research Article "COVID-19 induces a hyperactive phenotype in circulating platelets" in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have made the required changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for supporting Open Access publishing. We look forward to publishing your paper in PLOS Biology. 

Sincerely, 

Paula

---

Paula Jauregui, PhD 

Senior Editor 

PLOS Biology

pjaureguionieva@plos.org

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .