Persisting Endothelial Cell Activation and Hypercoagulability after COVID-19 Recovery—The Prospective Observational ROADMAP-Post COVID-19 Study

Round 1

Reviewer 1 Report

The authors perform a prospective observational study of endothelial and hypercoagulability markers on COVID-19 survivors who were primarily recruited for convalescent plasma donation. They conclude that there is activation of endothelial cells, in vivo thrombin generation and fibrin lysis up to 2 months from COVID-19 symptom onset.

 

Major Comments

1. Study showed overall decrease in von Willebrand Factor in subjects (median value 46%) compared with controls (113%) -this is different from what previous publications have mentioned.

The authors did not state in their discussion, the reasons for a depressed von Willebrand Factor (vWF) level in the subjects in the study. In contrast, both Forgaty et al (reference xxvii) and Townsend et al (reference xxviii) reported elevated endothelial markers including raised vWF levels. Forgaty showed that plasma levels of von Willebrand factor (VWF), factor VIII (FVIII), and soluble thrombomodulin were elevated at a median of 68 days after initial COVID-19 symptom resolution or discharge from the hospital.

Authors should discuss this difference in their revision.

Additional studies which are key to this discussion that focus on endothelial dysfunction/hypercoagulability will be the following :

Reference 1: von Meijenfeldt FA, Havervall S, Adelmeijer J, et al. Sustained prothrombotic changes in COVID-19 patients 4 months after hospital discharge. Blood Adv. 2021;5(3):756-759. doi:10.1182/bloodadvances.2020003968

Reference 2: Chioh FW, Fong SW, Young BE, et al. Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation. Elife. 2021;10:e64909. Published 2021 Mar 23. doi:10.7554/eLife.64909

 

2. Authors did not state clearly the demographic and clinical data of the subjects versus the controls.

The authors should incorporate a Table (Table 1) comparing the demographics of age, sex, blood group, existing co-morbidities, as well as the clinical data (i.e. time from diagnosis of COVID-19)) of both subjects and controls.

3. Study methodology has significant limitations

• One criteria which has significant ramifications to the study results was the requirement of normal PT, APTT and fibrinogen levels at the day of inclusion
• This is particularly key as in Townsend’s study (Figure 1) – there were a small subgroup of patients with elevated PT and elevated fibrinogen levels. Exclusion of such subjects will result in an unintended selection bias, where patients with hyperfibrinogenemia would have a hypercoagulable state.
• The study only included COVID-19 patients who were primarily recruited for plasma donation for convalescent plasma- where survivors with known cardiovascular disease were excluded from the study, and where they had meet general criteria for blood donation
• What was the requirement for blood donation, was there a criterion for donors not to be on any long term medications (as blood donations in some countries require donors to not be on medication)? Can we presume these would then select for a healthier subgroup of patients?
• Several questions remain unanswered including the state of hypercoagulability and endothelial activation in survivors with existing cardiovascular disease (would these markers be even more elevated)
• Authors claim (line 283-285) that “our data allow to propose the sensitivity of endothelial cells for SARS-CoV-2 or cytokine storm in COVID-19…result in long lasting endotheliopathy and enhanced in-vivo thrombin generation” – this is inaccurate as while all subjects had been exposed to SARS-CoV-2, cytokine storm in COVID-19 occur in a minority of patients. Given that clinical data such as severity of COVID-19 at diagnosis is unavailable, the authors cannot make this hasty generalization as they are unable to correlate extent of severity (mild/moderate/severe COVID-19) with the degree of post recovery endothelial activation and/or hypercoagulability.

 

4. Authors state that ROADMAP post COVID-19 study offers a comprehensive analysis of a large panel of biomarkers for endothelial cell activation and hypercoagulablilty.

• The current panel of Procoag-PPL, heparinase, TM, TFPI, vWF, D-dimer and FM, while providing interesting data, is not exactly comprehensive. Other key endothelial markers that are commonly used are ICAM, PCAM, P-selectin. For hypercoagulability, thrombin generation would be preferred rather than surrogate markers such as D-dimer and FM. Could the authors rationalize why these markers were preferred over global haemostatic tests such as thrombin generation?

 

5. Authors should discuss the clinical significance of the long-term persistence of endothelial activation/hypercoagulability. This includes thrombotic events post COVID-19.

Reference 1: Fan BE, Cheung C. Post COVID-19 Arterial Thromboembolism: A Clear and Present Danger [published online ahead of print, 2021 Apr 15]. Semin Thromb Hemost. 2021;10.1055/s-0041-1728717. doi:10.1055/s-0041-1728717

Reference 2: Engelen MM, Vandenbriele C, Balthazar T, et al. Venous Thromboembolism in Patients Discharged after COVID-19 Hospitalization. Semin Thromb Hemost. 2021;47(4):362-371. doi:10.1055/s-0041-1727284

Minor Comment:

1. Multiple spelling mistakes and grammatical errors

Line 27 “cellular hypercoagulability” – suggest remove “cellular”

Line 29 “role of hypercoagulability in long-COVID merits to be investigated” should be “role of hypercoagulability in long-COVID merits investigation”

Line 38 “SARAS-CoV-2” should be “SARS-CoV-2”

Line 55 “aalmost five million patients” –“aalmost” is spelt wrongly. This should be amended to “more than” as the number of deaths due to COVID-19 now exceeds 5.3 million.

Line 254 “extacellular matrix” should be “extracellular matrix”

Author Response

1. Study showed overall decrease in von Willebrand Factor in subjects (median value 46%) compared with controls (113%) -this is different from what previous publications have mentioned.

The authors did not state in their discussion, the reasons for a depressed von Willebrand Factor (vWF) level in the subjects in the study. In contrast, both Forgaty et al (reference xxvii) and Townsend et al (reference xxviii) reported elevated endothelial markers including raised vWF levels. Forgaty showed that plasma levels of von Willebrand factor (VWF), factor VIII (FVIII), and soluble thrombomodulin were elevated at a median of 68 days after initial COVID-19 symptom resolution or discharge from the hospital.

Authors should discuss this difference in their revision.

Additional studies which are key to this discussion that focus on endothelial dysfunction/hypercoagulability will be the following :

Reference 1: von Meijenfeldt FA, Havervall S, Adelmeijer J, et al. Sustained prothrombotic changes in COVID-19 patients 4 months after hospital discharge. Blood Adv. 2021;5(3):756-759. doi:10.1182/bloodadvances.2020003968

Reference 2: Chioh FW, Fong SW, Young BE, et al. Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation. Elife. 2021;10:e64909. Published 2021 Mar 23. doi:10.7554/eLife.64909

We wish to thank you for your comment. Indeed, there is a discrepancy between our findings regarding FvW and those reported by the studies that you mentioned. A possible explanation might be the prevalence of the blood group O among the survivors. Nevertheless, our study is underpowered to explore this hypothesis. Thus, a bias effect cannot be excluded. To avoid misleading conclusions and confusion in the literature we decided to omit the data on FvW from the present article.

1. Authors did not state clearly the demographic and clinical data of the subjects versus the controls.

The authors should incorporate a Table (Table 1) comparing the demographics of age, sex, blood group, existing co-morbidities, as well as the clinical data (i.e. time from diagnosis of COVID-19)) of both subjects and controls.

As you recommended, we introduced the Table 1 which summarizes the demographics and clinical data of the survivors and controls. There are no comorbidities because these were among the exclusion criteria since survivors were selected for the phase III clinical trial on the efficacy and safety of convalescence plasmatherapy.

1. Study methodology has significant limitations

• One criteria which has significant ramifications to the study results was the requirement of normal PT, APTT and fibrinogen levels at the day of inclusion
• This is particularly key as in Townsend’s study (Figure 1) – there were a small subgroup of patients with elevated PT and elevated fibrinogen levels. Exclusion of such subjects will result in an unintended selection bias, where patients with hyperfibrinogenemia would have a hypercoagulable state.
• The study only included COVID-19 patients who were primarily recruited for plasma donation for convalescent plasma- where survivors with known cardiovascular disease were excluded from the study, and where they had meet general criteria for blood donation
• What was the requirement for blood donation, was there a criterion for donors not to be on any long term medications (as blood donations in some countries require donors to not be on medication)? Can we presume these would then select for a healthier subgroup of patients?
• Several questions remain unanswered including the state of hypercoagulability and endothelial activation in survivors with existing cardiovascular disease (would these markers be even more elevated)
• Authors claim (line 283-285) that “our data allow to propose the sensitivity of endothelial cells for SARS-CoV-2 or cytokine storm in COVID-19…result in long lasting endotheliopathy and enhanced in-vivo thrombin generation” – this is inaccurate as while all subjects had been exposed to SARS-CoV-2, cytokine storm in COVID-19 occur in a minority of patients. Given that clinical data such as severity of COVID-19 at diagnosis is unavailable, the authors cannot make this hasty generalization as they are unable to correlate extent of severity (mild/moderate/severe COVID-19) with the degree of post recovery endothelial activation and/or hypercoagulability.

 We agree with your remark that the studied cohort of COVID-19 survivors represents a selected population with absence of underlying disease (mainly cardiovascular) which are related with endothelial cell activation prior to SARS-CoV-2 infection as well as with the absence of blood coagulation abnormalities. Thus, survivors of the studied cohort represent COVID-19 patients who did not present risk factors for disease worsening and also had a less severe disease trajectory. According to your comment we enrich the discussion by analysing this point. We also raise the question of the severety and the frequency of endothelial cell activation in survivors with risk factors of disease severity. (lines 435- 459)

1. Authors state that ROADMAP post COVID-19 study offers a comprehensive analysis of a large panel of biomarkers for endothelial cell activation and hypercoagulablilty.

• The current panel of Procoag-PPL, heparinase, TM, TFPI, vWF, D-dimer and FM, while providing interesting data, is not exactly comprehensive. Other key endothelial markers that are commonly used are ICAM, PCAM, P-selectin. For hypercoagulability, thrombin generation would be preferred rather than surrogate markers such as D-dimer and FM. Could the authors rationalize why these markers were preferred over global haemostatic tests such as thrombin generation?

As you recommended, we change the sentence by omitting the word “comprehensive”. Regarding your comment on the selection of biomarkers: We measured soluble TM, and TFPI because both are biomarkers of endothelial cell activation which also interfere in thrombin generation process (TM via the protein C/Protein S system is involved in the downregulation process and TFPI prolongs the lag-time of thrombin generation). Since our laboratory has long experience in the study of thrombin generation, we are more confident in the assessment of these biomarkers. In contrast, our experience in the measurement of ICAM and PCAM is limited and for this reason we did not include these biomarkers in the panel. in the measurement  in downregulates the propagation. P-selectin and thrombin generation (assessed with Thrombinoscope assay) were not assessed because of limited quantity of plasma. Nevertheless, we believe that it is not necessary to mention this problematic in the article.

 

1. Authors should discuss the clinical significance of the long-term persistence of endothelial activation/hypercoagulability. This includes thrombotic events post COVID-19.

Reference 1: Fan BE, Cheung C. Post COVID-19 Arterial Thromboembolism: A Clear and Present Danger [published online ahead of print, 2021 Apr 15]. Semin Thromb Hemost. 2021;10.1055/s-0041-1728717. doi:10.1055/s-0041-1728717

Reference 2: Engelen MM, Vandenbriele C, Balthazar T, et al. Venous Thromboembolism in Patients Discharged after COVID-19 Hospitalization. Semin Thromb Hemost. 2021;47(4):362-371. doi:10.1055/s-0041-1727284

 

We added a relevant paragraph in the discussion session (lines 507-515) and we added the references that you suggested

 

Minor Comment:

1. Multiple spelling mistakes and grammatical errors

Line 27 “cellular hypercoagulability” – suggest remove “cellular”

Line 29 “role of hypercoagulability in long-COVID merits to be investigated” should be “role of hypercoagulability in long-COVID merits investigation”

Line 38 “SARAS-CoV-2” should be “SARS-CoV-2”

Line 55 “aalmost five million patients” –“aalmost” is spelt wrongly. This should be amended to “more than” as the number of deaths due to COVID-19 now exceeds 5.3 million.

Line 254 “extacellular matrix” should be “extracellular matrix”

We did the relevant modifications and we did our best correcte the typing and grammar errors

Reviewer 2 Report

Dear Editor

Dear Authors,

Thank you very much for the opportunity to review this interesting manuscript. This is a nicely written paper. I would like to suggest to address the following points in order to improve the quality of the manuscript.

 

 

1. Abstract:

-please change "un" into "an" in the line 32.

 

2. Introduction:

-Aims are described.

please change " aalmost" into " almost" in the line 55.

3. Methods

-Ethical approval was included

4. Results:

-Is the severity of the COVID-19 a risk factor for endothelial cell activation and hypercoagulability after recovery? Please include the description of the disease severity of these survivors at diagnosis. 

Author Response

1. Abstract:

-please change "un" into "an" in the line 32.

We did the corrections

1. Introduction:

-Aims are described.

please change " aalmost" into " almost" in the line 55.

1. Methods

-Ethical approval was included

1. Results:

-Is the severity of the COVID-19 a risk factor for endothelial cell activation and hypercoagulability after recovery? Please include the description of the disease severity of these survivors at diagnosis. 

The majority of the patients had mild COVID-19. However, as we mention in the Discussion due to the continuously hard epidemiological situation in Greece and the permanent saturation of the hospitals our colleagues there are absorbed with the treatment of the hospitalized patients and they do not have the adequate time or the human resources in order to thoroughly analyze the clinical data of the patients in order to provide a comprehensive clinical profile of the patients which will allow an unbised multivariate analysis to identify the risk factors for persistent hypercoagulability and endothelial cell activation. For this reason, and in order to avoid misleading conclusions we did not present the available clinical data in details and we did not perform the relevant statistical analysis. As soon as the actual pandemic wave will be controlled we will perform further analysis of the clinic-biological data.

R3

The following changes to the manuscript are required before possible consideration of publication:
1. the manuscript needs to be checked by a native speaker.

We did our best in order to improve the language of the manuscript

2. citations do not comply with the editorial requirements.

An editing of the References has been performed

3. in the introduction, the authors emphasize that hypercoagulability and endothelial damage accompany COVID-19, especially in severe form. Nevertheless, the authors must mention that in addition to hypercoagulability, fibrinolytic unbalance is also a key component of COVID-19 (DOI: 10.1055/a-1346-3178).

As you recommended, we added the fibrinolytic unbalance (line 67) and the reference that you proposed (Ref3)

4. I have no comments on the qualification of patients and the control group. However, the authors must compare demographic data between both groups in the results. 

We added the Table 1 which summarizes and compares demographics of the two groups

5. the authors provided CVs for the tests, but please add the detection limits for each laboratory parameter.

The detection limits for each assay is zero, and we added this information in the Materials and Methods section (line 152)

6. I am not convinced that the evaluation of blood groups is related to the subject of the study.

We preferred to keep this information (that blood group is not associated with persistent activation of endothelial cells and hypercoagulability) because some studies have shown that blood group is associated with the disease worsening risk.

7. table 1 is not understandable - the authors give the mean and median, the study group size and p-value are missing. What does "% frequency of alterations" mean?

Former Table 1 is Table 2 in the revised version. We did the necessary changes.

8. mL, not ml.

We did the corrections

9. the abbreviations in the table should be explained below it.

We added the explanations of the abreviations in Table 2 (former Table 1)

10. what do the values in bold mean in table 2?

Former Table 1 is Table 2 in the revised version. We did the necessary changes.

11. in the discussion, the authors must emphasize what is new in their studies and what distinguishes their research from others.
12. the limitations are described in a relatively trivial way.

We re-wrote the discussion in order to better demonstrate its originality and to place our findings and the limitations of the study into the actual discussion in the literature

 

13. literature is prepared contrary to the requirements of the journal. 

We did the editing of the References

 

Reviewer 3 Report

The following changes to the manuscript are required before possible consideration of publication:
1. the manuscript needs to be checked by a native speaker.
2. citations do not comply with the editorial requirements.
3. in the introduction, the authors emphasize that hypercoagulability and endothelial damage accompany COVID-19, especially in severe form. Nevertheless, the authors must mention that in addition to hypercoagulability, fibrinolytic unbalance is also a key component of COVID-19 (DOI: 10.1055/a-1346-3178).
4. I have no comments on the qualification of patients and the control group. However, the authors must compare demographic data between both groups in the results. 
5. the authors provided CVs for the tests, but please add the detection limits for each laboratory parameter.
6. I am not convinced that the evaluation of blood groups is related to the subject of the study.
7. table 1 is not understandable - the authors give the mean and median, the study group size and p-value are missing. What does "% frequency of alterations" mean?
8. mL, not ml.
9. the abbreviations in the table should be explained below it.
10. what do the values in bold mean in table 2?
11. in the discussion, the authors must emphasize what is new in their studies and what distinguishes their research from others.
12. the limitations are described in a relatively trivial way.
13. literature is prepared contrary to the requirements of the journal. 

Author Response

The following changes to the manuscript are required before possible consideration of publication:
1. the manuscript needs to be checked by a native speaker.

We did our best in order to improve the language of the manuscript

2. citations do not comply with the editorial requirements.

An editing of the References has been performed

3. in the introduction, the authors emphasize that hypercoagulability and endothelial damage accompany COVID-19, especially in severe form. Nevertheless, the authors must mention that in addition to hypercoagulability, fibrinolytic unbalance is also a key component of COVID-19 (DOI: 10.1055/a-1346-3178).

As you recommended, we added the fibrinolytic unbalance (line 67) and the reference that you proposed (Ref3)

4. I have no comments on the qualification of patients and the control group. However, the authors must compare demographic data between both groups in the results. 

We added the Table 1 which summarizes and compares demographics of the two groups

5. the authors provided CVs for the tests, but please add the detection limits for each laboratory parameter.

The detection limits for each assay is zero, and we added this information in the Materials and Methods section (line 152)

6. I am not convinced that the evaluation of blood groups is related to the subject of the study.

We preferred to keep this information (that blood group is not associated with persistent activation of endothelial cells and hypercoagulability) because some studies have shown that blood group is associated with the disease worsening risk.

7. table 1 is not understandable - the authors give the mean and median, the study group size and p-value are missing. What does "% frequency of alterations" mean?

Former Table 1 is Table 2 in the revised version. We did the necessary changes.

8. mL, not ml.

We did the corrections

9. the abbreviations in the table should be explained below it.

We added the explanations of the abreviations in Table 2 (former Table 1)

10. what do the values in bold mean in table 2?

Former Table 1 is Table 2 in the revised version. We did the necessary changes.

11. in the discussion, the authors must emphasize what is new in their studies and what distinguishes their research from others.
12. the limitations are described in a relatively trivial way.

We re-wrote the discussion in order to better demonstrate its originality and to place our findings and the limitations of the study into the actual discussion in the literature

 

13. literature is prepared contrary to the requirements of the journal. 

We did the editing of the References

 

Round 2

Reviewer 1 Report

The manuscript is significantly improved, however, the points below need addressing:

Major point:

1. Limitations of study not fully addressed: 

The authors should mention the previous point highlighted:

A) Selection criteria of normal PT/APTT and fibrinogen excludes a subset of survivors with a persistent hypercoagulable state. 

• One criteria which has significant ramifications to the study results was the requirement of normal PT, APTT and fibrinogen levels at the day of inclusion
• This is particularly key as in Townsend’s study (Figure 1) – there were a small subgroup of patients with elevated PT and elevated fibrinogen levels. Exclusion of such subjects will result in an unintended selection bias, where patients with hyperfibrinogenemia would have a hypercoagulable state.
• The study only included COVID-19 patients who were primarily recruited for plasma donation for convalescent plasma- where survivors with known cardiovascular disease were excluded from the study, and where they had meet general criteria for blood donation. 

B) This is a single point prevalence assessment of endothelial and coagulation parameters in COVID-19 survivors (who had no significant cardiovascular risk factors). There is no baseline endothelial or coagulation profiling performed during the survivors acute SARS-CoV-2 infection. The authors assume there is persisting endothelial activation based on the presumption that in healthy individuals, there is no baseline increase in endothelial cell activation and hypercoagulability- however, there is no longitudinal testing of these parameters to prove this. (hence how does one conclude that there is persisting endothelial cell activation and hypercoagulability in their title and discussion??)

C) In their limitations, authors should discuss that ideally, profiling should be performed during infection and after infection to prove causality of SARS-CoV-2 causing persistent endothelial cell activation and hypercoagulability (obtaining baseline bloods pre-infection is usually almost impossible). 

Minor points:

Authors write in lines 287 to 288 "Furthermore the roadMAP post covid-19 study showed that 8% of the survivors and PPL-ct shorter than the LNL... hypercoagulability persisting for at least 2 months after COVID-19 is a rather frequent phenomenon. 

• 8% survivors having persistent hypercoagulability is a low percentage and the word "frequent" here is inappropriate. However, this is still a significant percentage in otherwise previously well survivors.

Spelling error in line 312 "endothelia" should be "endothelial" 

It is highly disappointing that the authors had removed von Willebrand levels from the study and were reluctant to discuss their findings, just because they were unable to justify the difference in existing publications (Townsend, von Meijenfeldt etc.). Could it possibly be that the other endothelial markers (heparanase, sTM and TFPI) were much more sensitive markers of endothelial activation than vWF (which would be high-normal) - in this curated group of survivors who were well, did not have significant comorbidities and fit enough for convalescent plasma donation; as well as what the authors mentioned:  a significantly higher proportion of blood group O patients who will have a decreased baseline vWF level). I would encourage the authors try to add in their findings with reasonable justifications. 

Overall, the authors have performed a study which will be of great interest and relevance to the understanding of Long COVID pathophysiology.

I recommend major revisions (as above) before acceptance. 

Author Response

 

Major point:

1. Limitations of study not fully addressed: 

The authors should mention the previous point highlighted:

1. A) Selection criteria of normal PT/APTT and fibrinogen excludes a subset of survivors with a persistent hypercoagulable state. 

• One criteria which has significant ramifications to the study results was the requirement of normal PT, APTT and fibrinogen levels at the day of inclusion
• This is particularly key as in Townsend’s study (Figure 1) – there were a small subgroup of patients with elevated PT and elevated fibrinogen levels. Exclusion of such subjects will result in an unintended selection bias, where patients with hyperfibrinogenemia would have a hypercoagulable state.
• The study only included COVID-19 patients who were primarily recruited for plasma donation for convalescent plasma- where survivors with known cardiovascular disease were excluded from the study, and where they had meet general criteria for blood donation. 

Following your comment we added the following paragraph (lines 350-355): “In addition, the study enrolled a highly selected population of COVID-19 survivors (i.e. absence of underlying pathological conditions, normal clotting times and fibrinogen levels) and thus, excluded a subset of survivors with underlying conditions which are associated with hypercoagulable state or hyperfibrinogenemia. This, consequently, represents a selection bias resulting in underestimation of the frequency of persistent hypercoagulability and endothelial cell activation in COVID-19 survivors.» 

1. B) This is a single point prevalence assessment of endothelial and coagulation parameters in COVID-19 survivors (who had no significant cardiovascular risk factors). There is no baseline endothelial or coagulation profiling performed during the survivors acute SARS-CoV-2 infection. The authors assume there is persisting endothelial activation based on the presumption that in healthy individuals, there is no baseline increase in endothelial cell activation and hypercoagulability- however, there is no longitudinal testing of these parameters to prove this. (hence how does one conclude that there is persisting endothelial cell activation and hypercoagulability in their title and discussion??)

We added the following paragraph (lines 307-322) which we hope responds to your queries: “The ROADMAP-postCOVID-19 study, lucks a control group of survivors from other viral infections (non SARS-CoV-2) which provoke pneumonia or with other species of coronavirus. This is a common limitation of the studies which investigate immunological, vascular or hematological alterations is patients with COVId-19 and it is imposed by emergency conditions of the SARS-CoV-2 outbreak. Consequently, we cannot evaluate the degree of endothelial cell activation induced by SARS-CoV-2 infection as compared to other viruses. Similarly, it is not possible to evaluate if the endothelial cell activation is influenced by the type of the SARS-CoV-2 variant. Moreover, the present study was designed to assess the biomakrers of hypercoagulability and endothelial cell activation at a single point after recovery from COVID-19. Thus, it cannot provide any information on the kinetics of the hypercoagulable state upon infection by SRS-CoV-2 and manifestation of COVID-19. Furthermore, the absence of a baseline evaluation - prior or at the early phase of SARS-CoV-2 infection does not allow to evaluate the potential impact of pre-existing hypercoagulability or endothelial cell activation on the persistence and the intensity of the observed alterations of coagulation and endothelial cells in the survivors. »

We keep the term « persistent hypercoagulability and endothelial cell activation” because our data

1. C) In their limitations, authors should discuss that ideally, profiling should be performed during infection and after infection to prove causality of SARS-CoV-2 causing persistent endothelial cell activation and hypercoagulability (obtaining baseline bloods pre-infection is usually almost impossible). 

We agree with your comment and we added the following paragraph (lines 340 - 345) “To optimize the strategy for prompt identification of the patients at risk of vascular complications related with long-COVID-19 a longitudinal evaluation of the clinically relevant biomarkers of hypercoagulability and endothelial cell activation identified in the present study, could be performed. This strategy might be useful for a personalized risk assessment strategy and targeted treatment and has to be evaluated in a prospective clinical study. »

Minor points:

Authors write in lines 287 to 288 "Furthermore the roadMAP post covid-19 study showed that 8% of the survivors and PPL-ct shorter than the LNL... hypercoagulability persisting for at least 2 months after COVID-19 is a rather frequent phenomenon. 

• 8% survivors having persistent hypercoagulability is a low percentage and the word "frequent" here is inappropriate. However, this is still a significant percentage in otherwise previously well survivors.

We replaced « frequent » with “not rare”

Spelling error in line 312 "endothelia" should be "endothelial" 

It is highly disappointing that the authors had removed von Willebrand levels from the study and were reluctant to discuss their findings, just because they were unable to justify the difference in existing publications (Townsend, von Meijenfeldt etc.). Could it possibly be that the other endothelial markers (heparanase, sTM and TFPI) were much more sensitive markers of endothelial activation than vWF (which would be high-normal) - in this curated group of survivors who were well, did not have significant comorbidities and fit enough for convalescent plasma donation; as well as what the authors mentioned:  a significantly higher proportion of blood group O patients who will have a decreased baseline vWF level). I would encourage the authors try to add in their findings with reasonable justifications. 

Your first comment on this topic led us to be more « conservative » in the presentation of our data on FvW particularly regarding the risk to present a concept about FvW levels in survivors and the relationship with the bood group. So we feel more confident to do again the analysis in in a larger sample of COVID-19 survivors in order to confirm our hypothesis.

Reviewer 3 Report

The authors have addressed my concerns and improved the quality of the manuscript.

Author Response

Thank you

Round 3

Reviewer 1 Report

Manuscript is much improved, recommend accept in present form.