Reviewer ’s report  
Title: Thrombin and Factor Xa Link the Coagulation System with Liver Fibrosis  
 
Version: 2 Date: 22 Dec 2017  
 
Reviewer: Chris  Spek  
 
Reviewer's report:  
In the current paper the authors show that FXa inhibition limits experimental liver fibrosis 
whereas thrombin inhibition does not affect fibrosis. Next to the in vivo experiment, the authors 
perform in vitro experiments suggesting t hat both thrombin and FXa induce stellate cell 
activation. I am however not convinced by the data shown in Figure 1 based on which it is 
claimed that both FXa and thrombin induce SMA expression. The immuno pictures are not very 
convincing whereas on the mR NA level no differences are shown. The authors should 
substantiate their claim by including western blots with appropriate loading controls. Moreover, 
it is important to confirm the small differences on the mRNA level (in the FXa/thrombin 
combination exper iments) by including additional house keeping genes. Finally, it would be 
interesting to more extensively analyse stellate cell activation by studying collagen production 
(by Western blot) and proliferation.  
 
The in vivo study shows that FXa inhibition lim its liver fibrosis whereas thrombin inhibition 
does not seem to show any effect. This is potentially interesting and seems to be in line with the 
in vitro findings that only the FXa / thrombin combination is profibrotic in this setting. As such a 
claim is ad odds with previous literature showing that thrombin inhibition does limit liver 
fibrosis, it is essential to include data on the anticoagulant activity of the administered 
anticoagulants. Although it is claimed that FXa and thrombin inhibition results i n similar 
anticoagulant activity based on literature, it has to be shown in the current model to exclude the 
possibility that the observed differences are just due to different degrees of anticoagulation.  
 
The authors conclude that FXa inhibition may be a valuable new treatment option for liver 
fibrosis but is seems premature to make such a claim. Most importantly, treatment is started 
before the onset of fibrosis which does not mimic the clinical setting. Either include experiments 
in which treatment is de layed till after disease onset, or down tune the conclusion of the paper. 
The same holds true for the suggested involvement of PARs: A full page in the discussion (and a 
whole figure) is included to suggest the effect is PAR mediated but not a single exper iment using 
PAR activation/inhibition is included.  
 
Finally, it would be important to include control data to the figures. For instance, please include 
solvent controls in Figure 1I -K and 2E and add data of non fibrotic mice to figure 4.  
 
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.  
No 
Are the conclusions drawn adequately supporte d by the data shown?  
If not, please explain in your comments to the authors.  
No 
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  
 
I agree to the open peer review policy of the journal. I understa nd 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 confidential 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: 22 Dec 2017  
 
Reviewer: Chris  Spek  
 
Reviewer's report:  
In the current paper the authors show that FXa inhibition limits experimental liver fibrosis 
whereas thrombin inhibition does not affect fibrosis. Next to the in vivo experiment, the authors 
perform in vitro experiments suggesting t hat both thrombin and FXa induce stellate cell 
activation. I am however not convinced by the data shown in Figure 1 based on which it is 
claimed that both FXa and thrombin induce SMA expression. The immuno pictures are not very 
convincing whereas on the mR NA level no differences are shown. The authors should 
substantiate their claim by including western blots with appropriate loading controls. Moreover, 
it is important to confirm the small differences on the mRNA level (in the FXa/thrombin 
combination exper iments) by including additional house keeping genes. Finally, it would be 
interesting to more extensively analyse stellate cell activation by studying collagen production 
(by Western blot) and proliferation.  
 
The in vivo study shows that FXa inhibition lim its liver fibrosis whereas thrombin inhibition 
does not seem to show any effect. This is potentially interesting and seems to be in line with the 
in vitro findings that only the FXa / thrombin combination is profibrotic in this setting. As such a 
claim is ad odds with previous literature showing that thrombin inhibition does limit liver 
fibrosis, it is essential to include data on the anticoagulant activity of the administered 
anticoagulants. Although it is claimed that FXa and thrombin inhibition results i n similar 
anticoagulant activity based on literature, it has to be shown in the current model to exclude the 
possibility that the observed differences are just due to different degrees of anticoagulation.  
 
The authors conclude that FXa inhibition may be a valuable new treatment option for liver 
fibrosis but is seems premature to make such a claim. Most importantly, treatment is started 
before the onset of fibrosis which does not mimic the clinical setting. Either include experiments 
in which treatment is de layed till after disease onset, or down tune the conclusion of the paper. 
The same holds true for the suggested involvement of PARs: A full page in the discussion (and a 
whole figure) is included to suggest the effect is PAR mediated but not a single exper iment using 
PAR activation/inhibition is included.  
 
Finally, it would be important to include control data to the figures. For instance, please include 
solvent controls in Figure 1I -K and 2E and add data of non fibrotic mice to figure 4.  
 
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.  
No 
Are the conclusions drawn adequately supporte d by the data shown?  
If not, please explain in your comments to the authors.  
No 
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  
 
I agree to the open peer review policy of the journal. I understa nd 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 confidential 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: 22 Dec 2017  
 
Reviewer: Chris  Spek  
 
Reviewer's report:  
In the current paper the authors show that FXa inhibition limits experimental liver fibrosis 
whereas thrombin inhibition does not affect fibrosis. Next to the in vivo experiment, the authors 
perform in vitro experiments suggesting t hat both thrombin and FXa induce stellate cell 
activation. I am however not convinced by the data shown in Figure 1 based on which it is 
claimed that both FXa and thrombin induce SMA expression. The immuno pictures are not very 
convincing whereas on the mR NA level no differences are shown. The authors should 
substantiate their claim by including western blots with appropriate loading controls. Moreover, 
it is important to confirm the small differences on the mRNA level (in the FXa/thrombin 
combination exper iments) by including additional house keeping genes. Finally, it would be 
interesting to more extensively analyse stellate cell activation by studying collagen production 
(by Western blot) and proliferation.  
 
The in vivo study shows that FXa inhibition lim its liver fibrosis whereas thrombin inhibition 
does not seem to show any effect. This is potentially interesting and seems to be in line with the 
in vitro findings that only the FXa / thrombin combination is profibrotic in this setting. As such a 
claim is ad odds with previous literature showing that thrombin inhibition does limit liver 
fibrosis, it is essential to include data on the anticoagulant activity of the administered 
anticoagulants. Although it is claimed that FXa and thrombin inhibition results i n similar 
anticoagulant activity based on literature, it has to be shown in the current model to exclude the 
possibility that the observed differences are just due to different degrees of anticoagulation.  
 
The authors conclude that FXa inhibition may be a valuable new treatment option for liver 
fibrosis but is seems premature to make such a claim. Most importantly, treatment is started 
before the onset of fibrosis which does not mimic the clinical setting. Either include experiments 
in which treatment is de layed till after disease onset, or down tune the conclusion of the paper. 
The same holds true for the suggested involvement of PARs: A full page in the discussion (and a 
whole figure) is included to suggest the effect is PAR mediated but not a single exper iment using 
PAR activation/inhibition is included.  
 
Finally, it would be important to include control data to the figures. For instance, please include 
solvent controls in Figure 1I -K and 2E and add data of non fibrotic mice to figure 4.  
 
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.  
No 
Are the conclusions drawn adequately supporte d by the data shown?  
If not, please explain in your comments to the authors.  
No 
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  
 
I agree to the open peer review policy of the journal. I understa nd 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 confidential comments 
to the editors,  which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant