Clarification of fibrin generation and degradation reaction processes of clot-fibrinolysis waveform in hemorrhagic disorders

Tomoko Matsumoto; Nukumi Tujii; Daiki Shimomura; Aya Kouno; Takeshi Suzuki; Sho Shinohara; Nobuo Arai; Hiroshi Kurono; Osamu Kumano; Mikio Kamioka

doi:10.1371/journal.pone.0320111

Peer Review History

Original SubmissionFebruary 13, 2025
19 Mar 2025 Decision Letter - Tomasz Kaminski, Editor PONE-D-25-07516Clarification of fibrin generation and degradation reaction processes of clot-fibrinolysis waveform in hemorrhagic disordersPLOS ONE Dear Dr. Matsumoto, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 03 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Tomasz W. Kaminski Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: “This study was supported by JSPS KAKENHI (grant number: JP 21K09089).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We gratefully acknowledge the technical and interpretive skills of Hiroshi Masutani, Akihiro Nakamura, and Akira Kondo. This study was supported by JSPS KAKENHI (grant number: JP 21K09089).” We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “This study was supported by JSPS KAKENHI (grant number: JP 21K09089).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Competing Interests section: “The first author has no conflicts of interest. T. Suzuki, S. Shinohara, N. Arai, and H. Kurono are employees of Sysmex Corporation. O. Kumano was an employee of the Sysmex Corporation when the study was conducted.” Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 6. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 8. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 9. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Additional Editor Comments: Dear Authors, Thank you for your submission. After careful review, the feedback from the reviewers indicates that the paper is promising and aligns with the scope of PLOS ONE. However, there are several significant issues that need to be addressed before it can be considered for publication. We recommend that you proceed with revisions to address these concerns. We believe that with the necessary improvements, your manuscript will be in a much stronger position for acceptance. We look forward to receiving your revised manuscript. Best regards, Tomasz W Kaminski Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Comments to the Authors The aim of the present study is to determine whether transmittance changes in CFWA reflect coagulation and fibrinolysis reaction by comparing them with other methods. The authors also compared normal plasma with coagulation factor-deficient plasma samples in CFWA. This study provides a valuable contribution to evaluating CFWA as a clinical application using basic protein assays. 1. The authors used plasma samples provided by company. To detect statistical differences, all experiments were performed three times. However, the authors conducted three experiments using only one sample of each plasma, which suggests that the authors assessed the error within the experimental procedures. The authors should use different lot samples or as mentioned in the limitations, it would be better to use patient plasma. 2. The authors used rabbit anti-fibrinogen antibody provided by MP Biomedicals for immunoblotting. However, I could not find such an antibody on the MP Biomedicals web site. If the antibody was prepared by the authors, the authors should cite a reference that previously used this antibody. If this antibody is being reported for first time, the authors should provide additional data demonstrating its specificity for fibrinogen, including which part of fibrinogen it detects and so on, as this is critical for this study. 3. In the immunoblotting assay, some lanes appear to contain different sample amounts. The authors should include an internal control (e.g., transferrin) or, at the very least, stain the membrane with Coomassie or ponceau S to confirm equal loading. 4. In general, alteplase is used at concentration of approximately 10 -100 nM for in vitro clot lysis assays. Is the amount of r-tPA used in this experiment appropriate? 5. In addition to alpha2 antiplasmin, PAI-1 also play a role in fibrinolysis and should be briefly mentioned in the discussion section. 6. P14, lane 6-7, N.D., note detected, probably not detected? 7. P8, lane 22, FCM should be changed to FMC. The authors should confirm these detail again. Reviewer #2: In this manuscript, the authors compare the results of their already published assy, the clot-fibrinolysis waveform analysis (CFWA), that simultaneously evaluate coagulation and fibrinolysis in vitro, to the measurements of several specific proteins, relative markers of either coagulation or fibrinolysis. The paper address some interesting questions but is purely descriptive. Also, the writing could be improved to make it easier to read. I would have some major comments: 1/ For readers that are not initiated to CWA, it would be important to remind in the introduction that this global assay is performed on plasma and to compare it to whole blood global assays such as thromboelastography and to other plasma CLA (Pieters M, JTH 2019) or thrombin/plasmin generation fluorogenic assays. For example, why use kaolin as a trigger and not thrombin ? 2/ Page 3, line 19: to assess DOAC in vitro effects is different from assessing their clinical efficacy. Please reformulate. 3/ Figures 1A and B are redundant with 1C. Only 1C should be kept. 4/ Figure legends should be separated from the main text and put at the end of the manuscript 5/ Are PIC equivalent to PAP (plasmin-antiplasmin) complexes? If so, the name should be changed. 6/ It is unclear if all laboratory tests performed on CS-2400 analyzer included FDP, D-dimer and PIC. Also, the name of each commercial reagent kit should be added. 7/ The intriguing result from this study is the increase of FMC during the fibrinolysis phase, indicating that FMC are generated during fibrinolysis or that the antibody used to measure FM also recognize small fragments released from FM fibrinolysis... It would have been interesting to compare FMC to thrombin-antithrombin complexes that would really reflect thrombin generation overtime. 8/ Also fibrinolysis markers increase earlier in deficient-plasma compared to normal plasma due to potential less quality fibrin fibers: is there any way to analyse fibrin density on CS-analyzers (from OD value delta at different wavelenghts?) 9/ Results from Western Blot and Figure 4 description should be moved to the Result section. Also the meaning of the different FDP (HMW XDP, DD, Y, D and E fragments) should be explained in the Figure legend. 10/ The number of individual experiments or replicates should be specified at least in each figure/table legend. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0320111.r001
Revision 1
1 May 2025 Author Response Reviewer's Responses to Questions Comments to the Author Reviewer #1: Comments to the Authors The aim of the present study is to determine whether transmittance changes in CFWA reflect coagulation and fibrinolysis reaction by comparing them with other methods. The authors also compared normal plasma with coagulation factor-deficient plasma samples in CFWA. This study provides a valuable contribution to evaluating CFWA as a clinical application using basic protein assays. 1. The authors used plasma samples provided by company. To detect statistical differences, all experiments were performed three times. However, the authors conducted three experiments using only one sample of each plasma, which suggests that the authors assessed the error within the experimental procedures. The authors should use different lot samples or We would like to thank you for your comments. This article used commercially available plasma. We believe that the experiments necessary for investigating the tendency of CFWA were conducted using a single lot. Plasma deficient in factor VIII was tested in three other lots. The reason we selected factor VIII-deficient plasma is because of the severity of bleeding symptoms and the high proportion of patients with hemophilia A in congenital bleeding disorders. The results were similar to those reported in the paper. However, we agree with you that it would be a good idea to conduct further studies using plasma from other lots or patient plasma. We have added the following to the end of the discussion section. P.12; lane.34 “We will have to investigate the results using patient plasma in the future.” 2. The authors used rabbit anti-fibrinogen antibody provided by MP Biomedicals for immunoblotting. However, I could not find such an antibody on the MP Biomedicals web site. If the antibody was prepared by the authors, the authors should cite a reference that previously used this antibody. If this antibody is being reported for first time, the authors should provide additional data demonstrating its specificity for fibrinogen, including which part of fibrinogen it detects and so on, as this is critical for this study. We would like to thank you for your comments. The reagent was not labeled. P.6; lane 35 We added the label. This experiment used Polyclonal rabbit anti-human fibrinogen (A0080) from Cite Ab. 3. In the immunoblotting assay, some lanes appear to contain different sample amounts. The authors should include an internal control (e.g., transferrin) or, at the very least, stain the membrane with Coomassie or ponceau S to confirm equal loading. We would like to thank you for your comments. In the Immunoblot, we added the same amount of sample to all lanes. We measured the same marker as an internal control in each gel. Furthermore, when examining various deficient plasma, we always performed Immunoblot experiments using pool normal plasma as a control for each measurement. P.6; lane 29 “We added equal volumes to the gel.” 4. In general, alteplase is used at concentration of approximately 10 -100 nM for in vitro clot lysis assays. Is the amount of r-tPA used in this experiment appropriate? We would like to thank you for your comments. As you pointed out, the tPA concentration in CFWA is higher than the therapeutic concentration. CFWA is a method developed by Nogami, K., Matsumoto, T., et al. et al.: A novel simultaneous clot-fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in hemorrhagic disorders. Br J Haematol. 2019 Nov;187(4):518-529. doi: 10.1111/bjh.16111.　 This method reflects coagulation and fibrinolysis processes within 500 seconds, and can be measured quickly using an automatic analyzer. 5. In addition to alpha2 antiplasmin, PAI-1 also play a role in fibrinolysis and should be briefly mentioned in the discussion section. We would like to thank you for your comments. The following content has been added to the discussion. P.11; lane 10 “The participation of both α2-antiplasmin and PAI-I performs an important role in the fibrinolytic system. In this study, we did not examine a PAI-I fibrinolytic factors in detail. In the future, it is essential to investigate the relationship with CFWA using assays that reflect α2-antiplasmin and PAI-I.” 6. P14, lane 6-7, N.D., note detected, probably not detected? We have corrected the mistake. 7. P8, lane 22, FCM should be changed to FMC. The authors should confirm these detail again. We have corrected the mistake. Reviewer #2: In this manuscript, the authors compare the results of their already published assy, the clot-fibrinolysis waveform analysis (CFWA), that simultaneously evaluate coagulation and fibrinolysis in vitro, to the measurements of several specific proteins, relative markers of either coagulation or fibrinolysis. The paper address some interesting questions but is purely descriptive. Also, the writing could be improved to make it easier to read. I would have some major comments: 1/ For readers that are not initiated to CWA, it would be important to remind in the introduction that this global assay is performed on plasma and to compare it to whole blood global assays such as thromboelastography and to other plasma CLA (Pieters M, JTH 2019) or thrombin/plasmin generation fluorogenic assays. For example, why use kaolin as a trigger and not thrombin ? Page 3. line 13: “The thromboelastography can evaluate coagulation and fibrinolysis, but it has low reproducibility (Nagler M,et.al, T�H 2014). The clot lysis time (CLT) with thrombin addition can assess fibrinolysis, but it does not reflect intrinsic or extrinsic coagulation (Pieters M, et.al, JTH 2018). CFWA uses plasma as the measurement sample and an automated coagulation analyser, so results are available within 10 minutes and it has excellent reproducibility, and it can use plasma stored in a freezer. This investigation was performed using a modified APTT reagent to evaluate the bleeding tendency caused by a decrease in clotting factors, including haemophilia, which is an endogenous coagulation disorder.” We have added the above text and references to the Introduction. 2/ Page 3, line 19: to assess DOAC in vitro effects is different from assessing their clinical efficacy. Please reformulate. We would like to thank you for your comments. Page3, line 26; efficacy→in vitro effects We have made the changes you suggested. 3/ Figures 1A and B are redundant with 1C. Only 1C should be kept. We would like to thank you for your comments. We have combined Figure 1 into a single diagram. 4/ Figure legends should be separated from the main text and put at the end of the manuscript We would like to thank you for your comments. We have complied with the submission guidelines and prepared this paper, but we have moved the figure legend to the end of the main text as you suggested. 5/ Are PIC equivalent to PAP (plasmin-antiplasmin) complexes? If so, the name should be changed. We would like to thank you for your comments. As you pointed out, we changed the PIC to a PAP. 6/ It is unclear if all laboratory tests performed on CS-2400 analyzer included FDP, D-dimer and PIC. Also, the name of each commercial reagent kit should be added. We would like to thank you for your comments. We had already written this, but we have made it easier to understand. (Page 6, line 14-19) 7/ The intriguing result from this study is the increase of FMC during the fibrinolysis phase, indicating that FMC are generated during fibrinolysis or that the antibody used to measure FM also recognize small fragments released from FM fibrinolysis... It would have been interesting to compare FMC to thrombin-antithrombin complexes that would really reflect thrombin generation overtime. Your point is entirely correct. We have added it. The comparison between FMC and TAT is a subject for the next time. Page 9, line 24 “The increase in FMC in the fibrinolytic phase indicates that the antibodies used to measure FMC or to produce FMC recognize not only FMC but also small FMC fragments released from fibrinolysis.” We have added the above text to the results. 8/ Also fibrinolysis markers increase earlier in deficient-plasma compared to normal plasma due to potential less quality fibrin fibers: is there any way to analyse fibrin density on CS-analyzers (from OD value delta at different wavelenghts?) The automated coagulation analyser (CN-6000) reflected the fibrin density by measuring changes in turbidity of transmitted light. Reference 24 stated that when FVIII was added to factor VIII-deficient plasma (0-100 IU/dL), the transmitted light (�Transmittance) increased and the turbidity changes improved.　 This reference cites a report (Lilley L, et al. Res Pract Thromb Haemost. 2017;1:231-241) that found that the addition of FVIII increased the density of fibrin fibres, and that the fibrin clot fibres were composed of thinner, more highly branched fibres.　 We were able to analyse fibrin clots in the absence of clotting factors in detail in our study. Page 11, line 36 “Lilley L et al. demonstrated that the addition of FVIII increased the density of fibrin fibres, and that the fibrin clot fibres were composed of thinner, more highly branched fibres (25) In addition, in the present study, by analysing the fibrinolytic reaction, it was considered that in plasma deficient in clotting factors, the density of fibrin fibres decreased, and the fibres were thicker and did not branch as much.” The above text has been added to the discussion, and the references have also been added. 9/ Results from Western Blot and Figure 4 description should be moved to the Result section. Also the meaning of the different FDP (HMW XDP, DD, Y, D and E fragments) should be explained in the Figure legend. We would like to thank you for your comments. We have added an explanation of the molecular weight marker and FDP fragment fraction to Figure legend 4. 10/ The number of individual experiments or replicates should be specified at least in each figure/table legend. We would like to thank you for your comments. The number of times each item was examined has been added to the figure legend�n= �. Attachments Attachment Submitted filename: renamed_06fa8.docx https://doi.org/10.1371/journal.pone.0320111.r002
15 May 2025 Decision Letter - Tomasz Kaminski, Editor PONE-D-25-07516R1Clarification of fibrin generation and degradation reaction processes of clot-fibrinolysis waveform in hemorrhagic disordersPLOS ONE Dear Dr. Matsumoto, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 29 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Tomasz W. Kaminski Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Dear Authors, Thank you for your thorough and thoughtful revision of the manuscript. One reviewer has now recommended a minor revision, with remaining concerns leaning toward clarification rather than substantive change. The second reviewer did not respond to the re-invitation; however, based on my assessment, you have addressed most of their original comments. However, I believe that one point from the second reviewer’s earlier feedback still requires better attention: “The intriguing result from this study is the increase of FMC during the fibrinolysis phase, indicating that FMC are generated during fibrinolysis or that the antibody used to measure FM also recognize small fragments released from FM fibrinolysis... It would have been interesting to compare FMC to thrombin-antithrombin complexes that would really reflect thrombin generation over time.” This comment remains only partially addressed. Strengthening the discussion around this point, particularly regarding the interpretation of FMC levels and the potential value of TAT comparisons, would improve the clarity and impact of your findings. Given the overall quality of the revision and your responsiveness, I am recommending a decision of Minor Revision. Best regards, Tomasz W. Kaminski [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: As reviewer #1, I would like to point out some more things to the authors. In terms of question #2, the polyclonal anti-human fibrinogen antibody (A0080) is provided by Dako, not Cyte ab. It is likely that Cyte ab is the company which provided the data. And regarding #3, in some immunoblotting results, band intensities appeared to vary between lanes, despite equal loading of samples. To verify that the same amount of protein was loaded in each lane, immunoblotting for serum proteins such as albumin or transferrin should be performed. Alternatively, the transferred PVDF or nitrocellulose membrane should be stained with Ponceau S to assess total protein levels. Another option is to prepare duplicate gels, using one for immunoblotting and staining the other with Coomassie Brilliant Blue to confirm equal loading. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0320111.r003
Revision 2
11 Jun 2025 Author Response Manuscript ID: PONE-D-25-07516R1 Title: Clarification of fibrin generation and degradation reaction processes of clot-fibrinolysis waveform in hemorrhagic disorders We sincerely thank the Academic Editor and the reviewer for the thoughtful comments and suggestions regarding our revised manuscript. We have carefully addressed all remaining concerns and revised the manuscript accordingly. Below, we provide a point-by-point response to the comments. Editor’s Comment: The intriguing result from this study is the increase of FMC during the fibrinolysis phase, indicating that FMC are generated during fibrinolysis or that the antibody used to measure FM also recognize small fragments released from FM fibrinolysis... It would have been interesting to compare FMC to thrombin-antithrombin complexes that would really reflect thrombin generation over time. This comment remains only partially addressed. Strengthening the discussion around this point, particularly regarding the interpretation of FMC levels and the potential value of TAT comparisons, would improve the clarity and impact of your findings. Response: We appreciate this important suggestion and have revised the discussion section to better clarify the interpretation of increased FMC levels during the fibrinolysis phase. Specifically, we now note the following: Page 11, Lines 29-33�Thus, FMC may have the reactivity in fibrinolysis phase. It is also useful to measure thrombin-antithrombin complex (TAT) for more classification between coagulation and fibrinolysis phase reaction. Because TAT is considered to increase in only coagulation reaction. The relationship between TAT and FMC was reported in clinical study, which these markers showed similar tendency [24]. Another study to investigate the relationship is planned. We believe these additions have improved the clarity and scientific context of our findings. Although it is difficult to compare FMC tendency with that of TAT due to the lack of several samples, we would like to conduct it as another separated study. Again, thank you for your suggestion. Reviewer #1 Comments and Author Responses Comment 1: In terms of question #2, the polyclonal anti-human fibrinogen antibody (A0080) is provided by Dako, not Cyte ab. Response: Thank you for pointing this out. We have corrected the manuscript to properly cite the antibody as: Page 6, Lines 35–36� “Polyclonal rabbit anti-human fibrinogen ( Dako, Glostrup, Denmark)” Comment 2: In some immunoblotting results, band intensities appeared to vary between lanes, despite equal loading of samples. To verify that the same amount of protein was loaded in each lane, immunoblotting for serum proteins such as albumin or transferrin should be performed. Alternatively, the transferred PVDF or nitrocellulose membrane should be stained with Ponceau S to assess total protein levels. Another option is to prepare duplicate gels, using one for immunoblotting and staining the other with Coomassie Brilliant Blue to confirm equal loading. Response: We have taken the reviewer’s advice and performed the suggested verification. Specifically, we used the third approach: preparing duplicate gels, with one used for immunoblotting and the other for Coomassie Brilliant Blue (CBB) staining. This was conducted using FVIII-deficient plasma samples. Additionally, we measured the protein concentration at each point from 0 to 360 seconds using the BioSpec-nano (Shimazu-biotech) device with absorbance (280 nm) using the same sample as the immunoblot. The detailed methos were as follows. First, O.D. 280 was detected in BioSpec-nano in each sample. Second, the protein concentration was estimated from O.D. 280 in the analyzer. Coefficient of molar absorbance of human albumin was used for the calculation tentatively, and the tentative concentration was obtained. Third, the concentration was compared among the samples. The concentration of Lane 1 (Time 0) was defined as 100%. Each protein concentration value was shown as relative value against sample of Lane 1. Relative value (%) = Concentration of each sample / concentration of Lane 1 (Time 0) The range of protein concentration was 95.3-107.3% among 6 samples. The variability was within 10%, indicating that the protein concentration was the same level among the samples. Therefore, we suggested that the immunoblotting results reflected the change of fibrinogen/fibrin degradation. Furthermore, the CBB-stained gel showed comparable band volume values from 0 to 120 seconds, indicating consistent protein loading. After 180 seconds, a decrease in band volume was observed, which we attribute to the degradation of fibrin degradation product (FDP) fractions. These results support the accuracy of our immunoblotting data and the observed time-dependent changes in protein levels. For the detailed data, please find the “Immunoblot and CBB 1,2” files. We thank the reviewer and editor again for their guidance, which helped us strengthen our manuscript. Sincerely, Matsumoto Tomoko., Ph.D., Faculty of Health Care, Department of Clinical Laboratory Science, Tenri University, 80-1 Bessho-cho, Tenri, Nara 632-0018, Japan. Tel.: +81-743-63-7811; Fax.: +81-743-63-6211; E-mail: t.matsumoto@sta.tenri-u.ac.jp On behalf of all co-authors Attachments Attachment Submitted filename: renamed_73ce7.docx https://doi.org/10.1371/journal.pone.0320111.r004
16 Jun 2025 Decision Letter - Tomasz Kaminski, Editor Clarification of fibrin generation and degradation reaction processes of clot-fibrinolysis waveform in hemorrhagic disorders PONE-D-25-07516R2 Dear Dr. Matsumoto, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Tomasz W. Kaminski Academic Editor PLOS ONE Additional Editor Comments: Dear Authors, Thank you for your thorough revisions. After two rounds of review, I am pleased to confirm that all concerns have been satisfactorily addressed. The manuscript is now in excellent shape, and I have recommended it for acceptance. Congratulations on your work. Reviewers' comments: https://doi.org/10.1371/journal.pone.0320111.r005
Formally Accepted
Acceptance Letter - Tomasz Kaminski, Editor PONE-D-25-07516R2 PLOS ONE Dear Dr. Matsumoto, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Tomasz W. Kaminski Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0320111.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .