Reviewer ’s report  
Title: Thrombin and Factor Xa Link the Coagulation System with Liver Fibrosis  
 
Version: 2 Date: 01 Jan 2018  
 
Reviewer:  Thierry Hauet  
 
Reviewer's report:  
Please include all comments for the authors in this box rather than uploading your report as an 
attachment. Please only upload as attachments annotated versions of manuscripts, graphs, 
supporting materials or other aspects of your report which cannot be included in a text format.  
Please overwrite this text when adding your comments to the authors.  
 
In this paper, Dhar et al explore the effect of thrombin and factor Xa in vitro on hepatic stellate 
cells (HSC) activity and the  inhibition of these serine proteases in vivo in a mice model of 
hepatic fibrosis. They showed a synergetic effect of thrombin and factor Xa on HSC contractility 
and activation. Then, they demonstrated that the inhibition of factor Xa by rivaroxaban was ab le 
to reduce liver fibrosis in mice. This is very interesting but I have several remarks regarding this 
work:  
 
Methods:  
Doses of rivaroban and dabigtran used in the animal studies are very high compared to doses 
used in human therapeutics. How did these do ses were determined?  
INR is mentionned as the test used to evaluate the anticoagulant effect of each molecule. As the 
mechanisms of or rivaroxaban and dabigatran are different, their impact on INR is also very 
different and a similar result of INR does not  reflect a similar anticoagulant effect.  
Did you measure each molecule activity with specific tests?  
 
Results:  
In vitro studies:  
Factor Xa seems to have a stronger effet on α -SMA staining of LX2 cells than thrombin while 
the relative gene expression of α -SMA is not different between cells incubated with FXa or 
thrombin alone. Is it a representative image of the effect of factor Xa on α -SMA staining?  
Please compare relative gene expression in each group to untreated cells or clarify the way you 
express PCR results. Cells incubated with FXa or thrombin alone seem to have lower gene 
expression levels than untreated cells.  
Figure 2 legend: The first sentence is not very clear, have inhibitors been tested in this assay?  
In vivo studies:  
Please add data on the control group for fibrosis (no TAA).  
Table 1 does not provide additional informations compared to figure 4 except for the group 
numbers which can be added to figure 3 and 4 legends.  
Figure 3A legend: Only one image of magnification x 15 is showed (no x40 magnification).  
 
Discussion: Sentences on the independent effect of factor Xa and thrombin on HSC activation 
need to be moderated regarding the gene expression results compared to untreated cells.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately supporte d by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please spe cify what aspects require further 
assessment in your comments to the editors.  
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
Needs some language corrections before  being published  
Declaration of competing interests  
Please comp lete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of t his 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently apply ing for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests' below  
 
I agree to the open peer review  policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as con fidential comments 
to the editors, which will not be published.  
I agree to the open peer review policy of the journal  
Reviewer ’s report  
Title: Thrombin and Factor Xa Link the Coagulation System with Liver Fibrosis  
 
Version: 2 Date: 01 Jan 2018  
 
Reviewer:  Thierry Hauet  
 
Reviewer's report:  
Please include all comments for the authors in this box rather than uploading your report as an 
attachment. Please only upload as attachments annotated versions of manuscripts, graphs, 
supporting materials or other aspects of your report which cannot be included in a text format.  
Please overwrite this text when adding your comments to the authors.  
 
In this paper, Dhar et al explore the effect of thrombin and factor Xa in vitro on hepatic stellate 
cells (HSC) activity and the  inhibition of these serine proteases in vivo in a mice model of 
hepatic fibrosis. They showed a synergetic effect of thrombin and factor Xa on HSC contractility 
and activation. Then, they demonstrated that the inhibition of factor Xa by rivaroxaban was ab le 
to reduce liver fibrosis in mice. This is very interesting but I have several remarks regarding this 
work:  
 
Methods:  
Doses of rivaroban and dabigtran used in the animal studies are very high compared to doses 
used in human therapeutics. How did these do ses were determined?  
INR is mentionned as the test used to evaluate the anticoagulant effect of each molecule. As the 
mechanisms of or rivaroxaban and dabigatran are different, their impact on INR is also very 
different and a similar result of INR does not  reflect a similar anticoagulant effect.  
Did you measure each molecule activity with specific tests?  
 
Results:  
In vitro studies:  
Factor Xa seems to have a stronger effet on α -SMA staining of LX2 cells than thrombin while 
the relative gene expression of α -SMA is not different between cells incubated with FXa or 
thrombin alone. Is it a representative image of the effect of factor Xa on α -SMA staining?  
Please compare relative gene expression in each group to untreated cells or clarify the way you 
express PCR results. Cells incubated with FXa or thrombin alone seem to have lower gene 
expression levels than untreated cells.  
Figure 2 legend: The first sentence is not very clear, have inhibitors been tested in this assay?  
In vivo studies:  
Please add data on the control group for fibrosis (no TAA).  
Table 1 does not provide additional informations compared to figure 4 except for the group 
numbers which can be added to figure 3 and 4 legends.  
Figure 3A legend: Only one image of magnification x 15 is showed (no x40 magnification).  
 
Discussion: Sentences on the independent effect of factor Xa and thrombin on HSC activation 
need to be moderated regarding the gene expression results compared to untreated cells.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately supporte d by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please spe cify what aspects require further 
assessment in your comments to the editors.  
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
Needs some language corrections before  being published  
Declaration of competing interests  
Please comp lete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of t his 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently apply ing for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests' below  
 
I agree to the open peer review  policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as con fidential comments 
to the editors, which will not be published.  
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
Title: Thrombin and Factor Xa Link the Coagulation System with Liver Fibrosis  
 
Version: 2 Date: 01 Jan 2018  
 
Reviewer:  Thierry Hauet  
 
Reviewer's report:  
Please include all comments for the authors in this box rather than uploading your report as an 
attachment. Please only upload as attachments annotated versions of manuscripts, graphs, 
supporting materials or other aspects of your report which cannot be included in a text format.  
Please overwrite this text when adding your comments to the authors.  
 
In this paper, Dhar et al explore the effect of thrombin and factor Xa in vitro on hepatic stellate 
cells (HSC) activity and the  inhibition of these serine proteases in vivo in a mice model of 
hepatic fibrosis. They showed a synergetic effect of thrombin and factor Xa on HSC contractility 
and activation. Then, they demonstrated that the inhibition of factor Xa by rivaroxaban was ab le 
to reduce liver fibrosis in mice. This is very interesting but I have several remarks regarding this 
work:  
 
Methods:  
Doses of rivaroban and dabigtran used in the animal studies are very high compared to doses 
used in human therapeutics. How did these do ses were determined?  
INR is mentionned as the test used to evaluate the anticoagulant effect of each molecule. As the 
mechanisms of or rivaroxaban and dabigatran are different, their impact on INR is also very 
different and a similar result of INR does not  reflect a similar anticoagulant effect.  
Did you measure each molecule activity with specific tests?  
 
Results:  
In vitro studies:  
Factor Xa seems to have a stronger effet on α -SMA staining of LX2 cells than thrombin while 
the relative gene expression of α -SMA is not different between cells incubated with FXa or 
thrombin alone. Is it a representative image of the effect of factor Xa on α -SMA staining?  
Please compare relative gene expression in each group to untreated cells or clarify the way you 
express PCR results. Cells incubated with FXa or thrombin alone seem to have lower gene 
expression levels than untreated cells.  
Figure 2 legend: The first sentence is not very clear, have inhibitors been tested in this assay?  
In vivo studies:  
Please add data on the control group for fibrosis (no TAA).  
Table 1 does not provide additional informations compared to figure 4 except for the group 
numbers which can be added to figure 3 and 4 legends.  
Figure 3A legend: Only one image of magnification x 15 is showed (no x40 magnification).  
 
Discussion: Sentences on the independent effect of factor Xa and thrombin on HSC activation 
need to be moderated regarding the gene expression results compared to untreated cells.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
Yes 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusions drawn adequately supporte d by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please spe cify what aspects require further 
assessment in your comments to the editors.  
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
Needs some language corrections before  being published  
Declaration of competing interests  
Please comp lete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of t his 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
3. Do you hold or are you currently apply ing for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests' below  
 
I agree to the open peer review  policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as con fidential comments 
to the editors, which will not be published.  
I agree to the open peer review policy of the journal  
assistant