Review Reports

Round 1

Reviewer 1 Report

The article is well structured and easy to read.
All blocks of research are justified;
the research methodology is well thought out, a sufficient number of studies
have been carried out (in animals and with the participation of human
biomaterial). The results look convincing.
I recommend the article for publication.

Author Response

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits，I really appreciate it.

Reviewer 2 Report

Global comments:

This article is very sound and interesting with concern to the anti-aggregant mechanism of sanguinarine. The authors did a very elegant and complete study, using many different techniques, and their results and conclusions are well-supported by the study. Fist of all, congratulations for this ambitious and innovative study, which brings an interesting link between traditional medicine, with herbal drugs, and modern understanding and supportive data on the action mode.

Then, I have some comments on the study presentation and analysis, and on the methodology described. Methods are insufficiently described and sometimes not clear enough. The objective of each technical protocol must be clearly presented. For example, for healthy subjects, it is necessary to indicate how many were selected, and what is the gender distribution (M/F). It is not clear why these healthy subjects were recruited and included for the study. I understand that they were only platelet donors, out of any medication which could affect platelets for the past 2 weeks. This must be stated in material and methods at paragraph 2.4.

The authors report the sanguinarine concentrations sometimes in mg/ml and sometimes in µM. They should always use the same unit.

Mice are treated with 2 different dosages of sanguinarine (5.0 and 10.0 mg/kg). It would be necessary to indicate what is the kinetics course of the blood concentration for each sanguinarine dose. This will help to better analyse in-vivo and in-vitro results, and to compare data genberated on mice with the in-vitro experiments using the 3 different concentrations, expressed in µM.

After a dot ".", please leave always a space (to be verified in many parts of the text).

English language needs to me reviewed and some sentences must be corrected with the right wording. For example, "vehicle" should be replaced by "control". Many other words are inappropriately used throughout the text.

Specific comments:

For example in abstract, line 19, it should be "to immobilized fibrinogen" and not "in". Line 25 requires rewording. Line 26, the right wording of "indicate" is without "s". Line 27 "aberrant" is not appropriate; "excess of platelet activation" would be better. Many other similar approximations need attention in the text.

Page 2, lines 21-23: text presentation needs to be corrected.

Page 3, lines 4-5, "alteration" is not right; it should be "changes".

Page 3, line 13, "supplied" is not right; "introduced into" would be better.

Page 3, line 47: title of paragraph 2.6 must be corrected.

Page 4, paragraph 2.10, the authors should specify which parameters, markers, or proteins are tested with western-blot.

Page 5, line 1-22, English language must be revised.

On figures 2, 3, 4, 5, 6, and 7, "vehicle" must be replaced by "control".

Pages 6, 7 and figure 3 (and throughout the text), using the abbreviation of CRP for "Collagen Related Peptide" is confusing, as this abbreviation is usually assigned to "Cross Reacting Protein".

On figure 8, it would be useful to show the extra-cytoplasmic domain of FcγR...

Page 14: "Conclusion" must be a separate paragraph.

 

 

 

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Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Dan et al has described the platelet inhibitory activity of a benzophenanthridine alkaloid, sanguinarine, in this manuscript. Sanguinarine delayed occlusion of artery in a ferric chloride-induced thrombosis model in mice, while did not prolong bleeding time in a tail transection model. In an in vitro flow device model, sanguinarine reduced platelet adhesion and aggregation on collagen-coated surface. Platelet adhesion and spreading on fibrinogen coated surface, aggregation induced by collagen and collagen-related peptide (CRP), release of ATP from platelet, P-selectin expression on platelet, and binding of PAC-1 (a mAb) to platelet GPIIb/IIIa, were all attenuated by sanguinarine. Mechanism of action of sanguinarine appeared to be related to reduction of phosphorylation of proteins downstream of collagen receptor GPVI and fibrinogen receptor GPIIb/IIIa, and reduced intracellular Ca2+.

Overall this is a strong study that suggested possible antithrombotic (especially antiplatelet) effect and mechanism of sanguinarine. I have several comments that the authors should consider in a revised manuscript before publication:

1. P1 L15: ‘the formation of thrombosis’ is not quite right, should be ‘thrombus formation’ or simply ‘thrombosis’.
2. P1 L18 (and several other places throughout the manuscript): platelet release of what?
3. Fig 4: Panels b and d might have swapped places?
4. P8 L13: Sanguinarine at 2.5 micromolar does not appear to be inhibiting PLCγ2 according to Fig 5a & b despite the text stated otherwise.
5. Fig 5: Panels b and d appeared to be identical. I believe the graph correspond to panel c is not included.
6. P12 L13: Please use proper subscript for ferric chloride

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

The aim of the paper was to investigate the effects of sanguinarine on collagen-induced platelet functions. The authors studied effects of this drugs in vivo and in vitro. Sanguinarine was able to inhibit collagen-induced platelet aggregation, secretion, adhesion and spreading. The authors noticed an inhibition also in thrombus formation. In vivo. In conclusion, sanguinarine appeared to be a promising candidate for anti-platelet therapy.

How did the authors select Sanguinarine doses? Why did the authors give the mice multiple doses at different time points?

Did the authors check whether platelet aggregation was altered in Sanguinarine-treated mice versus control?

 

Which vehicle did the authors use?

Fig 4C: these graphs are confusing. I suggest to make one graph for each concentration? They can be on a column so it is easy to compared them anyway.

Did the authors check for changes in ADP-induced aggregation?

Fig. 8: it is confusing as it is still not clear which specific part of the pathway is inhibited by Sanguinarine.

 

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Thank you for considering all the comments and addressing clear and appropriate answers.

I just have 2 short additional comments.

The first one is for figure 8, for which the legend to figure is insufficiently informative. I think that it would be useful for readers to describe the impact points of sanguinarine on the scheme.

The second one concerns the half life of sanguinarine following its intake. I understand how the concentrations used for experiments were selected. However, as kinetics course cannot be shown, it would be useful to give an indication on the in-vivo half life, although this comment is not critical.

Author Response

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Author Response File: Author Response.docx