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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: In this manuscript, Matthew Holding and colleagues investigated the venom resistance of M. domestica. In different experiments, they examined whether the decreased binding between VWF and botrocetin results in functional resistance to coagulopathic effects, and whether M. domestica is capable of neutralizing venom components beyond CTLs.

I have several questions and remarks on the current version of this manuscript:

-Overall, it is nice data but the focus on VWF (as was expected from the title and abstract) is quickly lost, going to general resistance to coagulopathic effects. Therefore, I would suggest to change the title. In addition, often more explanation is needed at the start of a results paragraph, to explain why a certain experiment is performed.

For figure 1 and 2: “As expected, … platelet accumulation on the microfluidic surface was strongly inhibited by botrocetin and convulxin” (L135-136) Here, a short text is needed to explain why this should be expected. This is provided in the material and methods section so it could be removed there from both ‘Microfluidic Assays’ and ‘Aggregation Assays’ paragraphs, and be added to results section.

For figure 4, it is now not clear how this figure adds to the story (especially not with the current title/abstract), as the main research question here focusses on VWF, but in Figure 4 suddenly metalloproteinase inhibition is tested. Also, suddenly different venoms are used compared to Figure 3. The rationale for this should be provided in the results section.

-Figure 1: the authors conclude that opossum VWF requires a greater shear force to elongate. Is this significant? This is not indicated in the graph.

-Supplemental figure 1: from this figure, I’m not convinced with the conclusion that ‘there were no obvious differences in multimer distribution’. First, quantification (% to total) of high, intermediate and low molecular weight multimers is needed. Second, an equal concentration should be loaded on the gel to be able to compare, which seems not to be the case here.

-In Figure 2, images of fibrin formation under flow would be of added value.

-The human control is lacking in Figure 3, while it was nicely added in Figure 1 and 2. Regarding the lack of this comparison, the conclusion on L277-279 (Aspercetin appears to be able to function nearly as well in opossums as it does in humans in microfluidics assays (in both cases it has weak function), but not in whole blood assays) can’t be drawn as aspercetin was not tested here in whole blood assays using human whole blood.

-In the graphical abstract, aspercetin is mentioned as ‘not resistant’ in platelet adhesion, while convulxin and botrocetin are mentioned as ‘moderately resistant’. This is not correct when looking at Figure 2J-K?

Minor:

-The abbreviations CTL and SVMP are used in the introduction, however not written out fully when used for the first time. Please adjust this.

-“Concordant with the whole blood aggregometry analyses” (L145< L150) is mentioned in the results section of Figure 2. However, only microfluidic experiments using PRP are described thus far, and whole blood aggregometry experiments follow in Figure 3. This text should be adjusted.

-In the results section, refer to the specific subfigures (e.g. Figure 3B instead of Figure 3).

-Typo in the graphical abstract: sheer stress should be shear stress. Also, the shear stress mentioned here is 80 dyn/cm2 while 60 dyn/cm2 is mentioned in the figure legends.

Reviewer #2: This article discusses the resistance to envenomation of a small and poorly studied species of Opossum, focusing on coagulopathic effects. The article provides robust information by covering different types of coagulopathic activities to better understand the effects of resistance to envenomation, as well as using different species and isolated toxins, considering the animal ecology and its possible targets of resistance to envenomation. Therefore, I recommend the publication of the article, which brings new information about prey-predator coevolution (Arms race), contributing scientifically to a better understanding of the phenomenon.

Minor suggestions:

Please include an item in the Methodology section to describe statistical analysis used on the article

Line 59 – please include the description of CTLs here – once it´s the first time that the abbreviation is described

Line 300 – verifiy the reference

Sugegstion on figure 1: Do the AUC curves to statistically compare between Human and oposum shear stress could enrich the results.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:  Caroline Fabri Bittencourt Rodrigues

**********

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Dear Dr. Holding,

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Karen de Morais-Zani

Academic Editor

PLOS ONE

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Additional Editor Comments:

One of the reviewers suggests some minor corrections. We look forward to receiving your revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The authors have adequately and extensively addressed my comments. I have no further questions or comments.

Reviewer #2: The authors have satisfactorily addressed the issues raised in the first review. Only a few editing issues should be considered for submission.

Line 75 - different font from the rest of the text

on line 419, replace the term C-type lectins for SNACLECs.

line 455: italic missing on B. jararaca

line 557: missing parentesis

references with a different font from the rest of the text

duplicated references ( 49 to 51)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:  Caroline Fabri Bittencourt Rodrigues

**********

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Comparative microfluidic and enzymatic analyses reveal multifaceted snake venom resistance and novel VWF behavior in the opossum Monodelphis domestica

PONE-D-25-27609R2

Dear Dr. Holding,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Karen de Morais-Zani

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: