The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability

Round 1

Reviewer 1 Report

In this manuscript Joussaume et al described the mechanisms of Protein S involvement in endothelial cells permeability. They showed that Protein S increases both basal- and VEGFA- induced permeability of endothelial cell monolayers by activation p38 MAPK and RhoA/ROCK pathways. The manuscript is clear presented and experiments are good designed, however there are still some questions that can improve the presented manuscript.

Major

In the first paper (Blood 2012) from the same group the authors found that protein S inhibited VEGF-A–induced endothelial VEGFR2 phosphorylation and activation of mitogen-activated kinase-Erk1/2 and Akt and Protein S activated the tyrosine phosphatase SHP2. In the presented manuscript they only discuss activation of RhoA and P38. They also showed that protein S activate PAK which is the effector of Rac. It is well known that Rac is involved in the cytoskeleton remodeling, but this is also not discussed in the discussion part of the manuscript. Obviously protein S activate several pathways that are involved in regulation of endothelial cells permeability and this should be included in the discussion, not only RhoA and p38.

Minor

P.2, L 71. “activates HSP27 phosphorylation” Or phosphorylate, or activate, but can not activates HSP27 phosphorylation.

P. 2, L 84. “phosphorylates VEC phosphorylation” should be corrected.

Material and Methods. Or all materials should be included in the manuscript, or they all should be in the Supplements, but not part in the manuscript and other part in the supplementary table. I have no access to the Supplementary table 1.

P. 4, L.165. “20-30 μg of total protein lysate/lane”. How protein content was measured.

P. 4, L. 179 -180. “Equal amounts of protein in  Laemmli buffer were analyzed by SDS-PAGE” Equal amount could be analyzed by Ponceau S staining but not by SDS-PAGE.

P. 5, L. 205. were analysed using Fiji. Please describe what is Fiji?

P.6, L. 272. Prism 4.2 software. . -. Please correct.

P.6, L.276. “We found that PROS1 HSP27 phosphorylation occurred” Please correct this sentence.

P. 7, L. 283. “Human PROS1 regulates Vascular Endothelial Cadherin (VEC) phosphorylation” How it can regulate phosphorylation? Please correct.

P. 7, L. 288. “Indeed, skeletonization of images for VEC membrane staining” What is “skeletonization”, please correct or describe.

P.8, L.313. “of PAK-1 kinase on Ser144”. Better to include here activation of Rac and add in the discussion some sentences regarding Rac activation.

P. 11, L. 436. “by the sharp activation of HSP27” It is not activation, it is phosphorylation, or it should be presented a link between HSP27 phosphorylation and activation.

References should be corrected according to the journal stile.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

In this article entitled “The vitamin K-dependent anticoagulant factor, protein S, regulates vascular permeability.” the authors investigate the role for Protein S as an endogenous vascular permeabilizing factor. This is an interesting topic. The figures and tables are clear. Here are my comments:

1.     The abstract should be reorganized in sections background, materials and methods, results, and conclusion as it is presented as an original paper. The rest of the manuscript is also structured as an original paper and therefore it should be done for the abstract as well.

2.     The citations should be listed at the end of the manuscript only, whilst in the text it should be reported the progressive numbers linked to the cited article.

3.     The section introduction is very detailed and well written. It could be helpful for readers to provide a figure (or a diagram) summarizing the molecular pathways reported (it may also become the central figure of the paper).

4.     The first sentence of section discussion should be rephrased briefly reporting the results of the authors experiments. 

5.     Section discussion in general should be revised: the authors, for each paragraph, should discuss one of their results and put it in relation with previous published works. They, instead, reported in detail the results of other authors/papers and, therefore, the section discussion seems more a review than a conclusive part of an original paper. In conclusion, I think that the authors should give more space to their results, than citing other papers.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Per attached file.

Comments for author File: Comments.pdf

Numerous typos and grammatical issues.  Otherwise not bad.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors addressed the comments. Only the abstract still result unstructered and not organized in sections (backgrounds, matherials and methos, results and conclusion)

Fine

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Review of the submitted manuscript:  cimb-2920491.v2

The vitamin K-dependent anticoagulant factor, protein S, regulates vascular permeability

By:  Joussaume, A., et al.

Publication:  Curr.Issues Mol.Biol.

Summary:

            The authors revised this manuscript extensively.

Feedback: 

1)      Science:  The manuscript revisions improved it considerably.

2)      Presentation: 

a)      The authors use PROS1 as the acronym for Protein S.  Yet, sometimes in the table, figures or text other designations are used.  One cannot help but wonder if such other designations have some specific purpose.  Consistency would be helpful.

                    i.            Figures using ProS designation, at least some of the time, include: Figure 1, Figure 2, Figure 3, and Figure 4.

                  ii.            Table 1 uses ProS designation.

                iii.            ProS is used on line 374.

                iv.            ProS1 is used on line 498.

Author Response

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Author Response File: Author Response.pdf