Review Reports - Factor XII in Thrombosis and Thromboinflammation: From Molecular Biology to Clinical Translation

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

To the Authors:

This manuscript, entitled “Factor XII in Thrombosis and Thromboinflammation: From Molecular Biology to Clinical Translation,” provides a comprehensive review of the biological functions of factor XII (FXII) and the therapeutic potential of FXII inhibitors in thrombosis, inflammation, and hereditary angioedema. The topic is timely and relevant, as FXII inhibition represents a promising strategy for antithrombotic therapy with a potentially reduced bleeding risk. The manuscript effectively summarizes molecular mechanisms, experimental evidence, and emerging therapeutic approaches, including monoclonal antibodies and other inhibitors. I consider this review paper to be significant; however, I have several suggestions to improve the manuscript before it can be accepted.

Major Comments:

(1) The methods section only briefly states that PubMed was searched using the keyword “factor XII inhibitors” for articles published between 1950 and 2025. However, the search strategy lacks sufficient methodological detail. The authors should clarify: (a) whether additional databases (e.g., Scopus, Web of Science, Embase) were searched; (b) the full list of search terms used; and (c) the specific inclusion and exclusion criteria.

(2) The authors mainly summarized existing studies but provided limited critical evaluation of the evidence. For example, most of the data presented for FXII inhibitors originate from animal models, yet the translational limitations are not sufficiently discussed. Potential safety concerns, such as long-term immunogenicity, off-target effects, or complement system interactions, are only briefly mentioned. The differences between FXI and FXII inhibition strategies in clinical development could be better analyzed. The authors should strengthen the discussion by critically evaluating the strengths, limitations, and clinical relevance of the available evidence.

Minor Comments:

(1) The manuscript contains several minor grammatical and stylistic issues. For example:

(a) “demonstratethat” → should be “demonstrate that”

(b) “FXII s a central initiator” → missing “is”

(2) In the Introduction, the authors should briefly explain why FXII deficiency does not cause bleeding, as this is central to the rationale for FXII inhibition as a therapeutic strategy.

(3) Figure legends should be expanded to better explain the molecular interactions illustrated in the diagrams.

Author Response

Dear Reviewer,

Thank you for your valuable comments. We have revised the manuscript accordingly.

We agree and have expanded the Methods section to better describe the search strategy, search terms, study classification, and exclusion criteria. We also clarified the additional targeted search for clinically relevant FXII inhibitors.

We agree and have strengthened the Conclusions by adding a more critical evaluation of the predominance of animal data, translational limitations, safety concerns, and the differences between FXI- and FXII-targeted strategies.

All indicated grammatical and stylistic issues have been corrected, and the manuscript has undergone additional language editing.

We added a brief explanation in the Introduction clarifying why congenital FXII deficiency is not associated with bleeding.

We expanded the figure legends to better explain the molecular interactions shown.

Thank you again for your constructive suggestions.

Sincerely,

Jan Stępnicki

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This review addresses Factor XII (FXII) is highly relevant to thrombosis and thromboinflammation because critical role in contact activation, coagulation amplification, kallikrein–kinin signaling, complement biology, and device-associated thrombosis. Below are some comments may help strengthen the manuscripts:

At the end of abstract, it would be better to add a summarize sentence to address the purpose of this review.
There are multiple typo and grammatical problems in the text which reduce the readability. Such as “plateles,” “complement casade,” “Factor XII (FXII) s a central initiator,” “typy I and II,” and “demonstratethat.” These errors should be corrected carefully, including figure legends and tables.
There should be linkage between text and figures, like which figure corresponds to which part of the context.
In the figures, the abbreviation should be annotated. Such as “HK, BK”
The tables contain visible formatting artifacts and inconsistent phrasing.

Table 1, in the column for species, there are breedings and reagents, the author should make them clear.

Table 2 has same issue, column indicates animal models and therapeutic agents, while the context are models and devices and agents, the authors should clarify this.

Author Response

Dear Reviewer,

Thank you for your insightful and constructive comments. We have carefully revised the manuscript accordingly.

We agree and have added a concluding sentence to the Abstract to better highlight the purpose and scope of this review.

All indicated errors (e.g., “plateles,” “complement casade,” “FXII s,” “typy I and II,” “demonstratethat”) have been corrected. The manuscript, including figures and tables, has undergone thorough language editing.

We would like to clarify that all figures are placed immediately after the corresponding paragraphs in the manuscript to maintain logical flow and readability.

We agree and have added clear definitions of abbreviations (e.g., HK, BK) in all figure legends.

Formatting artifacts and inconsistencies have been corrected across all tables.

We revised the column description to to ensure consistency with the table content.

Thank you again for your valuable suggestions, which have improved the clarity and quality of our manuscript.

Sincerely,
Jan Stępnicki

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Factor XII (FXII), also known as Hageman factor, is a liver-derived zymogen that circulates in the blood and acts as a pathophysiological mediator of thrombosis and inflammation rather than an essential element of normal hemostasis, as its deficiency does not lead to bleeding disorders. Its autoactivation due to binding to negatively charged surfaces – such as extracellular RNA, neutrophil extracellular traps (NETs), polyphosphates released by activated platelets, or elements of damaged blood vessels – leads to the activation of factor XI to FXIa, triggering the intrinsic coagulation cascade, which leads to fibrin formation and stabilization of the thrombus. Besides the role in coagulation, FXII contributes to the activation of the kallikrein-kinin system and the classic complement pathway. Recent studies indicated that zymogen FXII has roles in immune response, wound healing, and angiogenesis.
Type III Hereditary Angioedema (HAE) is associated with mutations in the F12 gene, which causes "gain of function" (increased FXIIa activity), leading to excessive bradykinin production and tissue swelling.
Because FXII deficiency protects against thrombosis without causing bleeding, it became a major target for developing safe anticoagulation and immune modulation.
This comprehensive review summarizes the physiology of FXII, its pathophysiological roles, and current therapeutic options for FXII inhibition.
The writing is fluent, easy to understand, and perfectly reflects current knowledge on the topic.

Comments:
- Figure 1 illustrates the connecting role of FXII in coagulation, contact activation, and inflammation. However, the individual activation cascades are difficult to follow; the arrows are too far from the names of the factors. The legend lacks a description of the meaning of the individual arrow types (continuous and dashed arrows with different colors and curvatures). The reciprocal activation of FXIII and kallikrein is not obvious from the figure.
- The references for the statements described between lines 227-231 are missing.
- Centering should be avoided in table cells, especially if they contain bulleted lists.
- In the third column of Table 1, the heading "species" is incorrect, as some rows of the column also include the therapeutic agent.
- Line 290 contains both the abbreviation ECMO and the full name, although this is not the first usage of the abbreviation ECMO. Please introduce the abbreviation at the site ot first usage and use it only for later occasions.
- In line 312, the explanation of the abbreviation Ir-CPI is missing.
- The third column of Table 2 does not list the therapeutic agent for all studies.

Author Response

Dear Reviewer,

Thank you for your careful and constructive comments. We have revised the manuscript accordingly.

We agree that the original figure required improvement. The layout has been revised to make the activation cascades easier to follow. We also expanded the legend to explain the meaning of the different arrow types and colors. In addition, the reciprocal activation between FXII and kallikrein is now shown more clearly in both the figure and the legend.

The appropriate references have now been added to support the mentioned statements.

We agree and have revised the tables to avoid centering within cells.

We changed the heading “species”, which was inaccurate.

We have corrected the text so that extracorporeal membrane oxygenation is written in full at first mention, and the abbreviation ECMO is used consistently thereafter.

We agree and have added the full term for Ir-CPI at its first appearance in the text.

We have supplemented the table with the studies mentioned in the text.

Thank you again for your valuable comments, which helped us improve the clarity and consistency of the manuscript.

Sincerely,
Jan Stępnicki

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review article is addressing Factor XII and its therapeutic potential in the treatment of thrombosis and cardiovascular disease. The manuscript provides a brief overview of the field, including historical summary on FXII-targeted treatments, and summarizes various FXII inhibitors ranging from in vitro/in vivo assessments to those approved for clinical use. While the draft is somehow informative, the structure could be improved for clarity and overall impact.

The current title is general, and dose not adequately describe the manuscript content, and should be more specific. Perhaps this is also reflecting the challenge with manuscript structure.

Clearer sectioning into history, molecular biology, experimental evidence, and clinical trials/applications would improve readability and logical flow. Several section titles are not sufficient informative, and probably further reflecting the lack of clear section divisions.

Section 4. Molecular biology of FXII. This section is giving a general description of the intrinsic coagulation cascade relying on older references. The potential role of cellular components in FXII-mediated coagulation is warranted in this section. It is also notable that tissue factor-induced pathway and the cross-talks between the extrinsic and intrinsic pathway is not mentioned. Interaction points with intrinsic pathway is nor mentioned. Including these aspects would provide a more comprehensive overview of FXII biology and it’s the broader interaction within the coagulation system.

Section 7. Clinical inhibitors. The focus on Ir-CPI and garadicimab is appropriate, but the omission of rHA-Infestin is questioned, particularly since the manuscript acknowledges its ongoing clinical development. Including rHA-Infestin would provide a more complete picture of clinically relevant FXII inhibitors. A summary table outlining the inhibitors and current stages of clinical development would greatly enhance clarity for readers. The manuscript states that Ir-CPI is undergoing a phase II clinical trial, citing a 2023 reference. It would be important to update the status as of 2025.

Section 8 and 9. In these sections the authors details on pathophysiological effects on ischemic stroke and brain trauma, and anti-thrombotic prophylaxis connected to extracorporeal circuits. However, their placement after the clinical section may disrupt the logical flow. Presenting these discussions before the clinical section could improve coherence.

Figure: Given the complexity of the coagulation system, with numerous factors and interactions, figures are essential for clarity. The single figure currently included is too simplistic and does not provide sufficient information to the coagulation system. A more comprehensive figure is recommended, illustrating both the intrinsic and extrinsic pathways.

Overall: The manuscript content is informative but needs to be re-structured for clarity, including improvements in title (specificity), structural clarity, updates in references and inclusion of clinically relevant inhibitors, and more detail to figure.

Author Response

We would like to express our sincere gratitude for the insightful and constructive comments provided. They have significantly contributed to improving the clarity, structure, and scientific value of our manuscript.

The title has been revised to more accurately reflect the scope and significance of the manuscript, emphasizing its most valuable conceptual contribution.

Section 4 has been substantially expanded to include a detailed discussion of the tissue factor–induced pathway and the cross-talk between the extrinsic and intrinsic coagulation pathways.

To improve logical flow and readability, the order of sections has been modified. Information concerning individual FXII inhibitors and their potential clinical implications is now presented after the relevant pathophysiological concepts (currently Sections 7 and 8). This restructuring has resulted in a clearer, more coherent progression from molecular mechanisms to therapeutic applications.

The manuscript now includes up-to-date references on the progress of Ir-CPI development, including data available as of 2025.
Furthermore, rHA-Infestin-4 has been incorporated as a separate subsection, acknowledging its importance and contribution to the field.

A new table comparing the characteristics, mechanisms of action, and developmental stage of the various FXII inhibitors has been added, enhancing clarity and facilitating comparison for readers.

The original schematic has been replaced with a more comprehensive figure illustrating both intrinsic and extrinsic pathways, their interaction points, and the role of FXII in thromboinflammatory processes.

We believe these revisions have substantially strengthened the manuscript. We thank the reviewer once again for the valuable feedback that helped improve the quality and clarity of our work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The author reviewed the role of FXII in modulating the intrinsic pathway and the kallikrein–kinin system, and the promise of FXII inhibition for treatment of ischemic stroke and HAE. Overall, the quality of this manuscript was poor.

Lack of reference: line 167~174 “prostate cancer cells were shown to initiate coagulation through a FXII- dependent mechanism”.

Line 192, prolonged duration of FXII ASO or mAbs poses challenges in situations requiring rapid reversal—such as active bleeding. This sentence is confusing, as FXII is not known to be required for normal hemostasis (bleeding arrest).

Line 250, the content of cycloSD6 reducing thrombotic complication in FeCl3 triggered arterial thrombosis has a wrong reference.

Line 275~278, the description for Ir-CPI is redundant, this content appeared at lines 199~209. Similar redundant content appeared at line 353.

Author Response

- “Prostate cancer cells were shown to initiate coagulation through a FXII-dependent mechanism” – the reference has been corrected to the appropriate source.

- “Prolonged duration of FXII ASO or mAbs poses challenges in situations requiring rapid reversal—such as active bleeding” – this passage has been removed from the manuscript.

- CycloSD6 – the paragraph discussing this inhibitor has been revised and updated for clarity and accuracy.

- Ir-CPI – restructuring of the manuscript has eliminated redundancies and improved the coherence of this section.

The title has been revised to more accurately reflect the scope and significance of the manuscript, emphasizing its most valuable conceptual contribution.

Section 4 has been substantially expanded to include a detailed discussion of the tissue factor–induced pathway and the cross-talk between the extrinsic and intrinsic coagulation pathways.

A new table comparing the characteristics, mechanisms of action, and developmental stage of the various FXII inhibitors has been added, enhancing clarity and facilitating comparison for readers.

We believe these revisions have substantially strengthened the manuscript. We thank the reviewer once again for the valuable feedback that helped improve the quality and clarity of our work.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript structure has been improved, and it is in this respect easier to read. However, since only the manuscript with track changes are included, it is still not easy to read. It is recommended to include also the clean version.

In the new parts, references seems not to be fully merged with old content, which makes the revision look incomplete. For the new TF section in particular, the one reference is not of relevance, which further rises questions to the solidity and the foundation . This is a review paper, and it is expected that authors are citing the most appropriate original studies or review articles on the addressed topic.

The new title is still not reflecting fully the content of the review. Inflammation is not really addressed but only mentioned in the context of bradykinin.

Author Response

We appreciate your observations and have addressed them carefully.

In the revised submission, we have included a clean version showing only the newly added modifications to ensure the manuscript is easy to follow.

We have updated the references related to tissue factor, replaced the insufficiently relevant citation, and expanded the TF–intrinsic pathway section to better reflect key original studies and recent reviews.

To ensure consistency between the title and the manuscript, we have selected a new title that focuses on the clinical implementation of FXII’s role in thrombosis and thromboinflammation, which better reflects the content and emphasis of the review.

We believe that these revisions substantially improve the clarity and focus of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have significantly modified the contents and structure of the manuscript.

A confusing part in figure1: why the arrows from FXIIa, prekallikrein and HK points to “contact system”?
The contents in table2 should be checked, under column head “ species” there were description of more than just animal species, but also many other mixed info, please modify.
In table 3, the mAb 14E11 binds to the apple 2 and 4 domains of FXI, but not FXI, therefore technically it is not a FXII inhibitor.

Author Response

Dear Reviewer,

Thank you for these additional comments. We have carefully addressed all of your suggestions in the revised manuscript.
The arrows originating from FXIIa, prekallikrein, and HK have been corrected. We removed the confusing arrows pointing toward the “contact system”.

We have revised the table to ensure that the column title accurately reflects its content.

To avoid any ambiguity, we have removed 14E11 from Table 3.

All suggested modifications have now been implemented, and we believe they improve the clarity and accuracy of the manuscript.

Author Response File: Author Response.pdf