Prothrombotic Tendency in the Shadow of Cancer: Hypercoagulability, Impaired Clot Contraction and Fibrinolysis in Colorectal Cancer and Gastric Cancer Patients Undergoing Chemotherapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the present manuscript, Aleksandrowicz et al., have addressed the importance of fibrinolysis, coagulation and related thrombosis in cancer patients after chemotherapy. Overall, the authors have taken good interest in discussing the various aspects of the thrombotic process and kinetics.

 

Although my concerns with this manuscript are as mentioned below.

General concept comments:

1. The manuscript, although provides insights into vital change monitored during different phases of chemotherapy treatment, lacks the functional protein biology behind the observed changes; or in other terms it could have been improvised by assessment of levels vs activity of various prothrombotic and pro-coagulative factors levels in the blood of the patients.

 

2. Alongside the changes in the platelet numbers, are there any changes in mean platelet volume?

Author Response

Comments 1: “The manuscript, although provides insights into vital change monitored during different phases of chemotherapy treatment, lacks the functional protein biology behind the observed changes; or in other terms it could have been improvised by assessment of levels vs activity of various prothrombotic and pro-coagulative factors levels in the blood of the patients”.

Response 1:  We thank the reviewer for this important comment. We agree that a more in-depth analysis of the functional biology of proteins involved in the observed prothrombotic and procoagulant changes would provide additional, valuable mechanistic information. However, the aim of this study was primarily to comprehensively assess changes in hemostasis in cancer patients receiving chemotherapy. This was a pilot study, limited by design to markers that could be routinely measured in patients to enable translation of the obtained results into clinical practice. Nevertheless, we agree that further studies assessing the functional activity and molecular mechanisms underlying the observed changes would be a valuable direction for future work, as is mentioned in the revised version of the “Discussion”.

Comments 2: Alongside the changes in the platelet numbers, are there any changes in mean platelet volume?

Response 2: Thank you for pointing this out. We have included an additional figure as panel 3b to show the changes in platelet volume. Despite the increase in platelet count during chemotherapy, we did not observe an increase in MPV, and in fact, it was even reduced. This is partially consistent with some literature reports, that have shown chemotherapy generally leads to a decrease in MPV, often in conjunction with a decrease in total platelet count [Detopoulou P, Panoutsopoulos, G.I.; Mantoglou, M.; Michailidis, P.; et al. Relation of Mean Platelet Volume (MPV) with Cancer: A Systematic Review with a Focus on Disease Outcome on Twelve Types of Cancer. Curr Oncol. 2023;30:3391-3420. doi: 10.3390/curroncol30030258.; Sakin, A., Secmeler, S., Arici, S. et al. Prognostic Significance of Mean Platelet Volume on Local Advanced Non-Small Cell Lung Cancer Managed with Chemoradiotherapy. Sci Rep 2019; 9, 3959]. Some studies indicate that large-volume blood platelets are more reactive than the small ones. This may partly explain our observations in cancer patients undergoing chemotherapy - despite an increase in platelet count we showed reduced clot contraction.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for giving me the opportunity to review “Prothrombotic tendency in the shadow of cancer: hypercoagulability, impaired clot contraction and fibrinolysis in colorectal cancer and gastric cancer patients undergoing chemotherapy.” The manuscript investigates a highly relevant research topic and the premise is interesting. The patient population is well-chosen and the inclusion of matched controls a major strength.

 

However, I have to recommend that the paper be rejected in its current form, as there are significant shortcomings and even few, but significant, errors in the discussion.

I hope the authors will rework the manuscript, as the findings are interesting and should be presented.

To that end, I have written my comments below:

 

MAJOR

The authors use a conventional ROTEM assay for analysis of evidence of hypercoagulability. As this assay is developed for bleeding diagnostics and tissue factor is added in great excess in the assay, it is not very sensitive to thrombotic tendency, as the authors themselves point out. Was it considered to use a modified ROTEM which is more sensitive to fibrinolysis such the one described in:

Larsen JB, Hvas CL, Hvas AM. Modified Rotational Thromboelastometry Protocol Using Tissue Plasminogen Activator for Detection of Hypofibrinolysis and Hyperfibrinolysis. Methods Mol Biol. 2023;2663:763-773. doi: 10.1007/978-1-0716-3175-1_51. PMID: 37204751.

 

The clot retraction analysis is performed with washed platelets in an environment that is very different from the physiological. While this is of course acceptable, the limitations of this should be clearly stated and the results possibly be given a little less weight than they are in the discussion. For instance, might the CCR be merely a proxy measurement for platelet count?

 

Regarding the choice of methods: It is curious that the authors do not perform thrombin generation assays. They have suitable material (both PPP and PRP plasma) and these assays are well-validated, developed specifically for evaluating hypercoagulability, and would supplement the clot analyses nicely. It should be addressed in the discussion why these assays were omitted.

 

A higher percentage (78%) of patients in the “intermediate-risk” colon cancer group had stage IV advanced cancer compared to only 31% in the high-risk gastric cancer group. Might this not lessen differences in CCR found between the groups (as an advanced cancer has an increased thromboembolic risk and might therefore be more active in the coagulation). I assume this is the reason for partitioning the patients in disease stage IV and II/III groups, but this decision is not addressed when discussing the limitations in study design.

 

Line 162: Is the finding that “the severity of hypercoagulability decreased after three months” surprising? Should the patients not be expected to return to “healthy” coagulation after their cancer is reduced with chemotherapy? Do you have data on cancer stage after chemotherapy, or data on whether the chemotherapy was definitive/considered curative?

 

Line 134: “The activation of fibrinolysis in CS IV cancer”. Here should be added a reference to substantiate this, or, if it is a speculation, this should be clarified.

 

The experiment reported in figure 4 I find somewhat dubious. While I understand the rationale for examining if chemotherapy affects platelets specifically , I believe the limitations should be stated more clearly (you do not examine indirect results of chemotherapy, the setup is on healthy blood containing no cancer cells, the in vitro setup is very far from physiological etc.) Furthermore, you should state in the introduction if this experiment fills a knowledge gap, as the risk of thrombosis during chemotherapy is well-established. Furthermore, this experiment is not adequately explained in the method section, for instance choice of concentration, use of coagulation initiators etc. Lastly, using a ROTEM machine (validated and calibrated for whole blood) on plasma makes the results very hard to interpret. A control with no chemotherapy should at the very least have been shown in the graph, or a clear reference to a published protocol should be made. I think you should consider removing this experiment from the manuscript if you cannot meet the above revision comments.

 

Line 231: You suddenly include new results in the discussion section. It is highly relevant to include data on thrombotic events, but this should be stated in the results section!

When do these thromboses (pulmonary embolism and deep venous thrombosis) occur in relation to your sampling? Are these patients among the most hypercoagulable compared to patients without a thrombosis? Do the patients continue in you study if they experience thrombosis (usually anticoagulant treatment would be initiated immediately in these patients. These drugs will interfere significantly with analyses!)

Similarly regardig bleeding events: What kind of major bleeding? Did they receive blood transfusions before sampling? Did they show signs of reduced clot formation in the ROTEM assay? How did you gather these clinical informations?

 

Line 253: You again include results in the discussion. These should be moved to Results.

 

Line 297: This is an incorrect statement. When receiving chemotherapy containing cisplatin for instance, thromboprohylaxis is standard care for most cancer types.

 

Generally, the discussion appears to overstate the results and is highly speculative. For instance line 201: “This study showed …” instead of “supports the hypothesis” or similar.

Line 206-207: “low effectiveness of conventional tests” is a divisive statement. ROTEM is indeed not very effective (likely because it was developed for bleeding) but thrombin generation assays, thrombin-antithrombin complex and prothrombin fragment 1+2 assays are highly useful for showing hypercoagulable states and may even be used to predict thrombosis in cancer populations.

 

The study certainly adds important knowledge, but is relatively small, performs unvalidated analyses and at the very least needs to be validated and replicated as well as proven to be clinically significant, before these result can affect patient treatment. There are few references supporting the claims, and incorrect statements regarding current cancer treatment, which is concerning.

 

MINOR

Introduction, line 61 “… dissolution”. It is unclear if the reference for this is also no. 7. As it is an important point, the reference should be clear.

 

The line 104 “There were no significant differences …” conflicts with the following “elevated platelet count in cancer patients (line 110). Please amend to clarify.

 

Line 129: The abbreviations “CS IV” and “CS II/III” are not defined before they are used. Also, the definition could be added in the text below Table 2.

 

Figure 2: Is the text under the figure regarding panel D “times required to initiate fibrinolysis” correct?

 

Figure 2: Selected markings of statistically different between first and second sample would be helpful.

 

Line 154-155: What were the p values of these findings?

 

Table 2: The use of bold text to indicate findings of hypercoagulability is helpful, but easy to confuse for statistically significant (bold*). Consider indicating this in some other manner ie. colour.

 

Figure 3: The text under the figure appears to be misplaced; the legends for the unfilled/dark/grey appear under figure B, not A.

 

Line 241: “additional measurements of clot contractility may allow …” Do you think this will give you more knowledge? A thrombin generation assay would be the next logical step to include as this is a much more validated analysis and would supplement your clot contractility analysis nicely.

Author Response

Comments 1: The authors use a conventional ROTEM assay for analysis of evidence of hypercoagulability. As this assay is developed for bleeding diagnostics and tissue factor is added in great excess in the assay, it is not very sensitive to thrombotic tendency, as the authors themselves point out. Was it considered to use a modified ROTEM which is more sensitive to fibrinolysis such the one described in:   Larsen JB, Hvas CL, Hvas AM. Modified Rotational Thromboelastometry Protocol Using Tissue Plasminogen Activator for Detection of Hypofibrinolysis and Hyperfibrinolysis. Methods Mol Biol. 2023;2663:763-773. doi: 10.1007/978-1-0716-3175-1_51.

Response 1: We thank the reviewer for this remark and bringing this article to our attention. In fact, during our research we used a modified rotational thromboelastometry protocol to detect changes in the coagulation and fibrinolysis profile. Based on our own observations and consistent with scientific references, we selected optimal concentrations of activators, i.e., tissue factor (TF) or tissue plasminogen activator (tPA), which were characterized by high repeatability within given measurement series. The applied modifications allowed the use of significantly reduced concentrations of individual reagents to achieve increased method sensitivity and more accurate assessment of both enhanced and inhibited coagulation and fibrinolysis kinetics. For example, a small addition of tissue factor was necessary to normalize clotting times (reducing the scatter of results). The aim was to achieve clotting parameters, in a relatively short time, to ensure comparable conditions (starting point) for ongoing fibrinolysis. Compared to conventional ROTEM tests (EXTEM), we used significantly lower activator concentrations (e.g. the TF concentration was 11 times lower). It should be noted that the clotting times obtained in our study were significantly longer (166-436 s) compared to the typical values ​​observed in the EXTEM tests (38-79 s). In the case of tPA, we tried to select concentrations that did not inhibit the kinetics of clot formation or reduce the maximum clot amplitude (MCF), while ensuring efficient fibrinolysis in the samples of blood collected from healthy volunteers. This allowed us to obtain relatively reproducible measurements performed on the same samples within 2-4 hours of blood collection. We have successfully used this prococol to assess coagulation and fibrinolysis parameters in patients with polycythemia vera [Rusak T, Piszcz J, Misztal T, Brańska-Januszewska J, Tomasiak M. Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheresis. Thromb Res. 2014;134(1):192-8] or asthma [Tomasiak-Lozowska MM, Misztal T, Rusak T, Branska-Januszewska J, Bodzenta-Lukaszyk A, Tomasiak M. Asthma is associated with reduced fibrinolytic activity, abnormal clot architecture, and decreased clot retraction rate. Allergy. 2017;72(2):314-319]. The use of similar activator concentrations in thromboelastometric assessment of fibrinolysis has been commonly described in studies by other authors [Kuiper GJ, Kleinegris MC, van Oerle R, Spronk HM, Lancé MD, Ten Cate H, Henskens YM. Validation of a modified thromboelastometry approach to detect changes in fibrinolytic activity. Thromb J. 2016;14:1; van de Berg TW, Hulshof AM, Nagy M, van Oerle R, Sels JW, van Bussel B, Ten Cate H, Henskens Y, Spronk HMH; Dutch Covid-19 and Thrombosis Coalition (DCTC). Suggestions for global coagulation assays for the assessment of COVID-19 associated hypercoagulability. Thromb Res. 2021; 201:84-89.] or in the article mentioned by the reviewer [Methods Mol Biol. 2023;2663:763-773], especially in relation to the tPA concentrations used. We acknowledge that the description of the methodology may have suggested the use of substantially higher activator concentrations. To avoid any misunderstanding, the description of ROTEM methodology has been revised, and the final concentrations of all reagents present in the analysed sample are now explicitly stated.

Comments 2: The clot retraction analysis is performed with washed platelets in an environment that is very different from the physiological. While this is of course acceptable, the limitations of this should be clearly stated and the results possibly be given a little less weight than they are in the discussion. For instance, might the CCR be merely a proxy measurement for platelet count?

Response 2:  We agree that the use of washed plates has certain limitations. The clot retraction was measured using whole blood, platelet-rich plasma, and washed platelets, which were added to a buffer containing appropriate concentrations of ions and glucose, and in the case of washed platelets, also fibrinogen and thrombin. Our observations showed that the highest clot contraction rate (CRR) was achieved using washed platelets, with slightly slower kinetics observed in platelet-rich plasma (PRP), and the slowest in whole blood. Due to the significant impact of erythrocytes on the kinetics of clot contraction, we employed a procedure involving the addition of precisely defined volume of whole blood to the medium with calcium chloride. This approach enabled the assessment of the kinetics of the clot retraction process under standardized conditions. The described method is routinely used to determine the kinetics of clot retraction in individual patients [Tomasiak-Lozowska MM, Misztal T, Rusak T, Branska-Januszewska J, Bodzenta-Lukaszyk A, Tomasiak M. Asthma is associated with reduced fibrinolytic activity, abnormal clot architecture, and decreased clot retraction rate. Allergy. 2017;72(2):314-319; Tomasiak-Łozowska M.M., Rusak T., Misztal T., Bodzenta-Łukaszyk A., Tomasiak M., Reduced clot retraction rate and altered platelet energy production in patients with asthma. J. Asthma. 2016;53:589–598; Rusak T, Piszcz J, Misztal T, Brańska-Januszewska J, Tomasiak M. Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheresis. Thromb Res. 2014 Jul;134(1):192-8.] and to assess the effect of selected substances on this process [Misztal T, Golaszewska A, Tomasiak-Lozowska MM, Iwanicka M, Marcinczyk N, Leszczynska A, Chabielska E, Rusak T. The myeloperoxidase product, hypochlorous acid, reduces thrombus formation under flow and attenuates clot retraction and fibrinolysis in human blood. Free Radic Biol Med. 2019 Sep;141:426-437]. Only the results obtained for kinetic measurements in whole blood were presented in this paper. They were considered to be the most appropriate for physiological conditions, as the share of erythrocytes was taken into account. Unfortunately, in such a case, the influence of platelet count and fibrinogen concentration on clot contraction kinetics must be considered, so efforts were made to exclude patients with very high and very low values ​​of these parameters.

Comments 3:  Regarding the choice of methods: It is curious that the authors do not perform thrombin generation assays. They have suitable material (both PPP and PRP plasma) and these assays are well-validated, developed specifically for evaluating hypercoagulability, and would supplement the clot analyses nicely. It should be addressed in the discussion why these assays were omitted.

Response 3:   We thank the reviewer for raising this point. We fully agree that thrombin generation assays (TGA) are excellent for assessing hypercoagulability and could be complementary to our research.  At the time of the study, the necessary equipment for thrombin generation assays was not available in our laboratory, so we chose to use thromboelastometry (ROTEM) because it allows for the assessment of the viscoelastic properties of whole blood (clotting time, speed, and strength), encompassing the kinetics of clot formation, clot growth, and susceptibility to fibrinolysis. TGA, on the other hand, measures the ability of plasma to generate thrombin (initiation, propagation, and endogenous thrombin potential), but in an environment where erythrocytes—the most abundant morphological elements in blood—are absent.

Interpretation based exclusively on ROTEM measurements appears to be clinically justified, as parameters such as maximum clot firmness (MCF) and clot strength (G) demonstrate the strongest correlation with the occurrence of thrombotic events in patients with malignancies. Moreover, in the work of Sørensen et al.  [Sørensen B, Johansen P, Christiansen K, Woelke M, Ingerslev J. Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. J Thromb Haemost. 2003;1(3):551-558] the derivatives of ROTEM values display a remarkable degree of similarity between endogenous thrombin potential and thromboelastography (TEG).  We acknowledge that the thrombin generation assay (TGA) provides detailed information on coagulation dynamics and may represent a valuable tool for validation of the present findings. However, the current study was designed as a pilot investigation conducted in a limited patient cohort. Future studies in larger populations will therefore consider the inclusion of TGA (especially that such technical capabilities are now available) to further substantiate the assessment of hypercoagulability. The absence of thrombin generation assay (TGA) assessment was identified as a limitation and explicitly discussed in the Discussion section of the paper.

Comments 4:  A higher percentage (78%) of patients in the “intermediate-risk” colon cancer group had stage IV advanced cancer compared to only 31% in the high-risk gastric cancer group. Might this not lessen differences in CCR found between the groups (as an advanced cancer has an increased thromboembolic risk and might therefore be more active in the coagulation). I assume this is the reason for partitioning the patients in disease stage IV and II/III groups, but this decision is not addressed when discussing the limitations in study design.

 Response 4:   We thank the Reviewer for this insightful comment. We agree that the unequal distribution of disease stage between the groups may have influenced the observed differences in CCR and thromboembolic activity. The present study was pilot in nature, and no predefined selection criteria based on disease severity were applied. Patients were excluded only if platelet counts were below 150´10⁹/L or above 450 ´10⁹/L, or if fibrinogen levels exceeded 6 g/L prior to enrolment, as values outside these ranges were found to significantly affect the course of clot contraction. The stratification of patients into stage IV and stage II/III disease was introduced to better characterize the course of fibrinolysis, which in several cases appeared either markedly increased or significantly delayed. Furthermore, given the role of clot cross-linking (reflected by maximal clot firmness) and ongoing clot contraction as protective mechanisms against excessive fibrinolysis, the kinetics of clot contraction were analysed accordingly.

 

Comments 5:  Line 162: Is the finding that “the severity of hypercoagulability decreased after three months” surprising? Should the patients not be expected to return to “healthy” coagulation after their cancer is reduced with chemotherapy? Do you have data on cancer stage after chemotherapy, or data on whether the chemotherapy was definitive/considered curative?

Response 5:    Patients enrolled in the study remained under the care of the Department of Oncology both during the study and during long-term follow-up after the study was completed. During follow-up, some patients experienced thromboembolic events (n=16), including pulmonary embolism (n = 6), deep vein thrombosis (n=7), and thrombotic complications associated with a vascular port (n = 3). These events mostly occurred later, after the study was completed. The observed thrombotic events may confirm the increased risk of hypercoagulability in cancer patients, which has been previously described in the literature. Based on the available data, it was not possible to clearly determine whether the occurrence of thrombotic events was related to cancer progression or to the treatment administered. Nevertheless, the obtained results suggest that chemotherapy may be a factor increasing the risk of developing a hypercoagulable state. An interesting observation was a partial reduction in the severity of hypercoagulability parameters after approximately three months of follow-up, which appears to be in contradiction to the reported cases of thrombotic events. This phenomenon may result from complex interactions between the course of the cancer, the body's response to treatment, and dynamic changes in the hemostasis system during chemotherapy.

Regarding the evaluation of chemotherapy efficacy, clinical outcomes included tumor regression (n=12), partial disease stabilization, as well as disease progression (n=8). In a proportion of patients, chemotherapy was maintained, whereas in selected cases treatment was discontinued due to deterioration of the patients’ general clinical status.

Comments 6:  Line 134: “The activation of fibrinolysis in CS IV cancer”. Here should be added a reference to substantiate this, or, if it is a speculation, this should be clarified.

Response 6:   We thank the reviewer for this comment. We agree that the statement required clarification. Therefore, the description in the text has been changed, and it is now based more on our observations than on data from literature. The fibrinolytic system is often activated in cancer patients and may affect cancer progression, metastasis, and patient survival. Clinical studies have shown that elevated plasma levels of uPA and soluble uPAR are associated with cancer progression and metastasis in prostate, breast, bladder, and colorectal cancers. In advanced cancer with metastases, an imbalance in the hemostasic system is often observed, in which the fibrinolytic system plays an important role both as a compensatory mechanism for excessive activation of coagulation and as a factor facilitating the migration of cancer cells [Hisada Y, Mackman N. Profibrinolytic Factors and Cancer Progression, Metastasis, and Survival. Arterioscler Thromb Vasc Biol. 2025;45(10):1732-1741].  In fact, both impaired fibrinolysis and its excessive activation have been found in oncological patients. In this paper, we wanted to highlight that increased fibrinolysis may be associated with disease progression, especially with the occurrence of metastases, which is characteristic for patients in stage IV

Comments 7:  The experiment reported in figure 4 I find somewhat dubious. While I understand the rationale for examining if chemotherapy affects platelets specifically , I believe the limitations should be stated more clearly (you do not examine indirect results of chemotherapy, the setup is on healthy blood containing no cancer cells, the in vitro setup is very far from physiological etc.) Furthermore, you should state in the introduction if this experiment fills a knowledge gap, as the risk of thrombosis during chemotherapy is well-established. Furthermore, this experiment is not adequately explained in the method section, for instance choice of concentration, use of coagulation initiators etc. Lastly, using a ROTEM machine (validated and calibrated for whole blood) on plasma makes the results very hard to interpret. A control with no chemotherapy should at the very least have been shown in the graph, or a clear reference to a published protocol should be made. I think you should consider removing this experiment from the manuscript if you cannot meet the above revision comments.

 Response 7:    We thank the reviewer for this valuable comment. In the revised version of the manuscript, the description of this experiment has been substantially improved. The initial measurements were performed using whole blood, however, under these conditions the effects of the investigated compounds were largely negligible. Given that the studied drugs may potentially modulate platelet function, we considered that the use of plasma rich plasma (PRP) and the elimination of erythrocytes would allow for a more precise assessment of whether these compounds exert any direct effects on platelets. Furthermore, as only a single-dose exposure was investigated, higher concentrations were applied to approximate the effects of prolonged exposure to the compounds. This rationale has now been clarified in the description of the results obtained.

Comments 8:  Line 231: You suddenly include new results in the discussion section. It is highly relevant to include data on thrombotic events, but this should be stated in the results section!

 Response 8:    We thank reviewer for their valuable feedback. We acknowledge that the presentation of data in the original manuscript required clarification on our part. In the revised manuscript, this error has been corrected, and the results are presented correctly in the “Results” section. We would like to clarify that the phrasing used in the previous version of the “Discussion” was intended to highlight the increased incidence of thrombotic events among our patients, similar to what is reported in the literature.

Comments 9:  When do these thromboses (pulmonary embolism and deep venous thrombosis) occur in relation to your sampling? Are these patients among the most hypercoagulable compared to patients without a thrombosis? Do the patients continue in you study if they experience thrombosis (usually anticoagulant treatment would be initiated immediately in these patients. These drugs will interfere significantly with analyses!)

 Response 9:    We thank the reviewer for this valuable comment. In the revised version of the manuscript, we have clarified that the thrombotic events referred to occurred after the completion of the study, and that the affected patients received appropriate thromboprophylaxis. Patients with a previous/documented history of thrombotic events before the study were excluded from our research.

Comments 10:  Similarly regardig bleeding events: What kind of major bleeding? Did they receive blood transfusions before sampling? Did they show signs of reduced clot formation in the ROTEM assay? How did you gather these clinical informations?

 Response 10:    In the studied cohort, two bleeding events were recorded, occurring 3–5 months after study initiation. A causal relationship could not be established, but because similar observations of bleeding in cancer patients have been reported in the literature, this was reported in the discussion. This section has been significantly modified in the revised version of the manuscript.

Comments 11:  Line 253: You again include results in the discussion. These should be moved to Results.

 Response 11:    We, agree.  We have, accordingly moved this information to the results section, as the reviewer suggested.

Comments 12:  Line 297: This is an incorrect statement. When receiving chemotherapy containing cisplatin for instance, thromboprohylaxis is standard care for most cancer types.

Response 12:    The sentence referring to effects of platin-based drug was removed in the modified version of manuscript.

 Comments 13: Generally, the discussion appears to overstate the results and is highly speculative. For instance line 201: “This study showed …” instead of “supports the hypothesis” or similar.

Response 13:    The discussion has been significantly modified to avoid any potential misunderstanding.

 Comments 14: Line 206-207: “low effectiveness of conventional tests” is a divisive statement. ROTEM is indeed not very effective (likely because it was developed for bleeding) but thrombin generation assays, thrombin-antithrombin complex and prothrombin fragment 1+2 assays are highly useful for showing hypercoagulable states and may even be used to predict thrombosis in cancer populations.

 Response 14:    We referred to conventional tests used in clinical laboratories for routine coagulation diagnostics (e.g. APTT, PT), which do not allow increased clotting tendency to be detected. However, we would like to point out that in using a technique such as ROTEM it is possible to detect more subtle changes and that these changes may prove useful in identifying hypercoagulable states in cancer patients compared to the healthy population. The methods indicated by the reviewer are indeed useful in the analysis of coagulation status and hypercoagulation detects. However, an important advantage of ROTEM compared to other tests used in coagulation analysis is the ability to perform analyses using whole blood rather than plasma. This allows for a more comprehensive assessment of the hemostatic process, as it reflects the overall interaction between plasma coagulation factors, platelets, and other blood components. In the revised version, the discussion has been improved to include that the TGA may also be an effective tool for detecting hypercoagulable states.

 Comments 15: The study certainly adds important knowledge, but is relatively small, performs unvalidated analyses and at the very least needs to be validated and replicated as well as proven to be clinically significant, before these result can affect patient treatment. There are few references supporting the claims, and incorrect statements regarding current cancer treatment, which is concerning.

Response 15:    We thank the reviewer for this thoughtful and important comment. We fully agree that the present study is limited by the relatively small sample size and that the analyses should be interpreted as a pilot study. Accordingly, we have revised the manuscript to more clearly acknowledge these limitations and to explicitly state that our findings require further research and validation. In response to the reviewer’s comment, we have revised the “Discussion” to emphasize that the applied assays are intended as research tools rather than validated clinical diagnostics.  We have also softened statements that could be interpreted as suggesting immediate impact on patient management.

 MINOR COMMENTS (of Reviewer 2)

Comments 16: Introduction, line 61 “… dissolution”. It is unclear if the reference for this is also no. 7. As it is an important point, the reference should be clear.

Response 16: We appreciate this substantive comment from the reviewer. Reference 7 has also been included after the sentence beginning in line 57, as the reviewer suggested. 

Comments 17: The line 104 “There were no significant differences …” conflicts with the following “elevated platelet count in cancer patients (line 110). Please amend to clarify.

 Response 17:  We agree, the  misleading sentence has been removed from the results.

Comments 18: Line 129: The abbreviations “CS IV” and “CS II/III” are not defined before they are used. Also, the definition could be added in the text below Table 2.

  Response 18:   The CS abbreviations have been defined in the text (line 129) and also the definition has been added in the description of Table 2, as the reviewer suggested.

Comments 19: Figure 2: Is the text under the figure regarding panel D “times required to initiate fibrinolysis” correct?

  Response 19:  We agree that the original wording could cause misunderstanding. Therefore, the figure description below the figure has been changed to “lysis onset time (LOT)” – meaning the time from the onset of clot formation to a 15 % reduction in clot amplitude (fibrin dissolution), and the imprecise statement has been removed.

 Comments 20: Figure 2: Selected markings of statistically different between first and second sample would be helpful.

Response 20:   We agree with the reviewer’s suggestion. Statistically significant differences between samples, determined using a paired non-parametric test (Wilcoxon signed-rank test), have now been indicated in Figure 2 to improve clarity.

Comments 21: Line 154-155: What were the p values of these findings?

Response 21:    In the revised version of the manuscript, statistically significant differences (p-values) are now indicated.

Comments 22: Table 2: The use of bold text to indicate findings of hypercoagulability is helpful, but easy to confuse for statistically significant (bold*). Consider indicating this in some other manner ie. colour.

 Response 22:   As suggested by the reviewer, the color red was introduced to highlight areas indicative of hypercoagulability.

Comments 23:  Figure 3: The text under the figure appears to be misplaced; the legends for the unfilled/dark/grey appear under figure B, not A.  

Response 23:   We would like to thank the reviewer for their valuable and accurate comment. The text below the figures has been modified accordingly to ensure clarity and prevent any possible misinterpretation.   

Comments 24:  Line 241: “additional measurements of clot contractility may allow …” Do you think this will give you more knowledge? A thrombin generation assay would be the next logical step to include as this is a much more validated analysis and would supplement your clot contractility analysis nicely.

Response 24:   We thank the reviewer for this insightful comment. We agree that thrombin generation assay (TGA) is a well-validated and informative method for assessing hypercoagulability and could be a valuable complementary approach. In the revised manuscript, we have clarified the clot contractility measure. A deeper insight into mechanisms regulating clot contractility may add to better understating of the pathophysiology of hypercoagulability in cancer patients and warrants future more detailed studies. The discussion has been revised accordingly to better reflect this complementary role and to avoid overstatement.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Thank you for your thorough answers to comments and questions!

The concerns I had have been sufficiently answered, and the revised manuscript has been significantly improved in both metod and discussion section. 

Furthermore, the neccesary inconsistancies have been amended satisfactorily.