Persistently High Platelet Factor 4 Levels in an Adolescent with Recurrent Late Thrombotic Complications after SARS-CoV-2 mRNA Vaccination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have explained all the clinical outcomes well, but its difficult to understand the timeline of interventions and appearance of disease. Please reframe the highlighted text in abstract for better understanding. 

It would be nice to include a table of all the clinical parameters tested along with there value and ref range.

It is also suggested to include the flow chart of time line of occurrence of DVT, interventions given and vaccination dose.

Comments for author File: Comments.pdf

Author Response

1. Authors have explained all the clinical outcomes well, but its difficult to understand the timeline of interventions and appearance of disease. Please reframe the highlighted text in abstract for better understanding.

 

 ［Response］

Thank you very much for your excellent suggestion.

We have changed two figures (Figure 1: Page 3 and Figure 2：Page 4) for better understanding of the timeline and disease course.

 

 

2. It would be nice to include a table of all the clinical parameters tested along with there value and ref range.

 

［Response］

Thank you very much for your excellent suggestion.

We have added reference values for coagulation, fibrinolysis, and antiphospholipid antibody syndrome disease status(Lines 68,69,70,71,72,73,93,94,100,162,164).

Blood data for finding the cause of thrombosis are also presented in a table (Page 2).

 

 

3. It is also suggested to include the flow chart of time line of occurrence of DVT, interventions given and vaccination dose.

 

［Response］

Thank you very much for your excellent suggestion.

We have changed two figures (Figure 1: Page3 and Figure 2: Page4) for better understanding of the timeline and disease course.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors report a case of a 6-year-old girl of pediatric antiphospholipid syndrome in which the level of anti-platelet factor 4 was persistently elevated after administration of a SARS-CoV-2 57mRNA vaccine (BNT162b2); Deep vein thrombosis developed 6 months after the second vaccination.
It is not very clear whether the age at which the patient was diagnosed was 6 or 5 years. You initially said she was 6, later on line 70 you said she was 5.
Line 70...you said "the patient was treated with warfarin to prevent recurrence for 6 years", but I think you meant that "the patient was treated for 6 years with warfarin to prevent recurrence".
Figure 2 is good, schematic, but I recommend you to add if you have figures from the patient's diagnostic imaging.
I recommend adding a separate paragraph with the conclusions.
Any study requires the informed consent of the individual/parents and considerations of ethical issues, and these must be approved by the Ethics Committee. You have not mentioned anything about this information in the article. You only mentioned informed consent at the end
The article presents 19 references, being up to date.

Author Response

1. It is not very clear whether the age at which the patient was diagnosed was 6 or 5 years. You initially said she was 6, later on line 70 you said she was 5.

 

[Response]

Thank you very much for your important comments.

The patient was 6 years old at diagnosis. We have corrected the description (Page 2, Line 77).

 

2. 
   Line 70...you said "the patient was treated with warfarin to prevent recurrence for 6 years", but I think you meant that "the patient was treated for 6 years with warfarin to prevent recurrence".

 

[Response]

Thank you very much for your helpful recommendations.

We have replaced the sentence (Page 2, Line 77-79) as follows:

“the patient was treated for 6 years with warfarin to prevent recurrence and with beraprost sodium (prostaglandin I2 analog) for 5 years.”

 

 

3. Figure 2 is good, schematic, but I recommend you to add if you have figures from the patient's diagnostic imaging.

 

［Response］

Thank you very much for your excellent suggestion.

We have included Figure 4 as an image of the patient's abdominal echo (Page 5). Figure 4 shows that the inferior vena cava obstructed by a thrombus after vaccination.

4. 
   I recommend adding a separate paragraph with the conclusions.
   [Response]

Thank you very much for your helpful recommendations.

A separate paragraph entitled "Conclusion" has been created (Page7 Line 266).

 

 

5. Any study requires the informed consent of the individual/parents and considerations of ethical issues, and these must be approved by the Ethics Committee. You have not mentioned anything about this information in the article. 

 

[Response]

Thank you very much for your important comments.

We have added text to Introduction (Page 2, Lines 59-61) as follows:

“Consent for publication was obtained from the patient and her mother. The study protocol was approved by the Ethics Committee of Toho University Omori Medical Center (No.2022-101).”

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article entitled “Persistently high platelet factorv4 levels in an adolescent with recurrent late thrombotic complications after SARS-CoV-2 mRNA vaccination” describe a case report regarding the occurrence of deep vein thrombosis (DVT) and pulmonary embolism (PE) in a patient affected by pediatric Antiphospholipid Syndrome (APS) on  second dose of anti-COVID-19 vaccine. Interestingly, the patient had an elevation of PF4 in the clinical history with a severe elevation  after the first dose of vaccine and the elevation persisted for 5 months. Anyway, the patient received the second dose of vaccine and after 6 months occurred DVT and the level of PF4 was elevated. This care report is well written and the information is important as it is a message for all specialists  in the filed of the vaccines. In relation to measurement of PF4 and β-TG the literature reports about the effect of heparin on the mobilization of PF4 from the binding sites on endothelial cells falsely increasing the levels of PF4 (Busch C Thromb. Res.1980; Dawes  Thromb. Res. 1978; Hottz, E.D. Blood 2020; Middleton EA Blood 2020). On this basis, I suggest specifying if the studied patient  is heparinized or non-heparinized and adding the references above. I think that this article is suitable for publication in a revised version.

Author Response

1. In relation to measurement of PF4 and β-TG the literature reports about the effect of heparin on the mobilization of PF4 from the binding sites on endothelial cells falsely increasing the levels of PF4 (Busch C Thromb. Res.1980; Dawes  Thromb. Res. 1978; Hottz, E.D. Blood 2020; Middleton EA Blood 2020). On this basis, I suggest specifying if the studied patient  is heparinized or non-heparinized and adding the references above.

 

[Response]

Thank you very much for your important comments.

We have added the following text to Case presentation (Lines 103-104, 125-126):

“At the time of PF4 measurement, no heparinization was performed (Figure 2).”

“No heparin treatment was administered during the PF4 measurement.”

We then cited and added text regarding the papers identified by reviewers as follows (Page 5, Lines 182-184).

“In relation to the measurement of PF4 and β-TG, the literature reports the effect of heparin on the mobilization of PF4 from binding sites on endothelial cells, falsely increasing PF4 levels [6]. However, this case is not heparinized.”

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This is an interesting case report of a patient with a diagnosis of pediatric antiphospholipid syndrome, thrombosis and elevated levels of PF4. The authors attribute the occurrence of the second thrombosis to the vaccination with mRNA Covid -19 vaccine that allegedly triggered high persistent plasma levels of PF4 as a marker of intravascular platelet activation. However, the link between these events is poorly established. There is no evidence presented for a causative effect, and the literature references cited supposedly supporting some of the conclusions are misleading. The interval between vaccination and thrombosis (6 months after the second vaccination) is too long to establish a causative association and because the levels of PF4 before vaccinations are not known, it’s hard to prove that the vaccination instigated such high levels of this platelet chemokine.

 

Specific comments:

1.        The abstract is a bit incoherent. Please describe the events in chronological order.

 

2.        Please add references to the sentence (Line 44-46) “Elevated plasma β-thromboglobulin (βTG) and PF4 levels indicate platelet activation and are useful for diagnosing thrombosis, examining thrombus formation risk, and determining the efficacy of thrombosis treatment”

 

3.        Please add reference values to all laboratory results. Most importantly, reference values for antiphospholipid antibodies vary a lot between laboratories and depend on the kind of the test used, so it is difficult to evaluate the significance of the values included.

 

4.        Please use the new 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria instead of the 2006 Sapporo criteria to establish the diagnosis of APS.

 

5.        The diagnosis of APS technically fulfils the new criteria, but the aCL antibody levels over time are borderline. Was the patient tested for other thrombophilia conditions, like Protein C or S deficiency, Factor V Leiden, antithrombin III deficiency, Type 2B vWD, etc? Was any genetic testing for mutations performed?

 

6.        It would be helpful to add a chart of the events and therapy similar to Figure 2 for the period between occurrence of the first DVT until vaccination. It is not clear if different medication was subsequent or concomitant. In addition, the age of the first DVT is described first as 6 years then as 5 years.

 

7.        Please add references to the sentence (line 86) “At that time, many cases of thrombosis caused SARS-CoV-2 mRNA vaccines had been reported.” I am aware only of rare case reports, which can hardly be characterized as “many cases” and might only be coincidental and not causative.

 

8.        Figure 1 is not consistent with the text. For instance, there is information about a PF4 value of 350 ng/ml that is not reflected in the figure.

 

9.        The authors’ conclusion, “The persistence of high PF4 levels from vaccine administration to DVT recurrence suggested APS exacerbation by the vaccine,” is not supported by the data. Did the titer of the antiphospholipid antibodies increase after the vaccination? Did new autoantibodies appear after vaccination? Was the patient tested for anti-PF4 antibodies? Were there other markers of intravascular platelet activation (e.g. sP-selectin or similar) present? If there are plasma samples still available, these tests should be performed.

 

10.  Please state clearly if the patient was free of any estrogen therapy and was not pregnant.

 

11.  The reference 3 for the statement (L147-148) “DVT is common in patients with trauma who have elevated PF4 and βTG levels, and elevated PF4 levels are correlated with an increased risk of thrombosis [3]” is misleading. The paper cited refers to PF4 and betaTBG mRNA microparticles as a biomarker for DVT, but the plasma levels of PF4 or betaTBG are not included in this paper.

 

12.  The authors seem to follow PF4 levels in many patients. Can they provide information if they noticed a similar increase of PF4 levels in other patients and if they established the cause or circumstances? Did they notice other cases of increased PF4 levels after Covid-19 vaccination?

 

13.  There is a clear difference between VITT after adenoviral vaccines and TTS after mRNA vaccination. The text implies that VITT is common after mRNA Covid -19 vaccination. This is not true, so please revise this part of the manuscript.

Comments on the Quality of English Language

The English is understandable, albeit sometimes cumbersome

Author Response

Specific comments:

1. The abstract is a bit incoherent. Please describe the events in chronological order.

 

 [Response]

Thank you very much for your helpful recommendations.

We have revised the abstract (Lines 18-22, 27-28) as follows.

“Herein, we present a 17-year-old female who had underlying antiphospholipid syndrome (APS) and developed deep vein thrombosis (DVT) 6 months after her second BNT162b2 vaccine dose. Although she had no family history of thrombosis, she had previously developed DVT at 6 years of age, with thrombus formation in the right common iliac vein and the inferior vena cava, along with concomitant left pulmonary infarction.”

 

“The vaccine was hypothesized to exacerbate the patient's APS by activating coagulation.”

 

2. Please add references to the sentence (Line 44-46) “Elevated plasma β-thromboglobulin (βTG) and PF4 levels indicate platelet activation and are useful for diagnosing thrombosis, examining thrombus formation risk, and determining the efficacy of thrombosis treatment”

 

 [Response]

Thank you very much for your helpful recommendations.

 

The reference to “Elevated plasma β-thromboglobulin (βTG) and PF4 levels indicate platelet activation and are useful for diagnosing thrombosis, examining thrombus formation risk, and determining the efficacy of thrombosis treatment” was found only in a Japanese book, so it was deleted.

 

We have revised the Introduction (Page 2, Lines 44-46) as follows.:

“Elevated plasma β-thromboglobulin (βTG) and PF4 levels indicate platelet activation, and PF4 and βTG levels are not often measured in routine clinical practice because they are often released in response to stimuli such as blood sampling.”

 

And, we have added text to introduction (Page 2, Lines 52-54) as follows:

“In addition, PF4 is elevated in antiphospholipid syndrome (APS) when platelet activity is increased. This suggests that PF4 may be a marker for DVT [3].”

 

 

3. Please add reference values to all laboratory results. Most importantly, reference values for antiphospholipid antibodies vary a lot between laboratories and depend on the kind of the test used, so it is difficult to evaluate the significance of the values included.

 

［Response］

Thank you very much for your excellent suggestion.

Reference values are provided for blood coagulation/fibrinolysis data (Lines 68,69,70,71,72,73,93,94,100,162,164).

 

4. Please use the new 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria instead of the 2006 Sapporo criteria to establish the diagnosis of APS.

 

[Response]

Thank you very much for your important comments.

We have added text to Case presentation (Page 3, Lines 95-99) as follows:

“At age 16, she was diagnosed with APS based on the 2006 Sapporo criteria by her rheumatologist and pediatrician [4]. The 2023 American College of Rheumatology (ACR)/European League of Rheumatology (EULAR) APS criteria recommends “macrovascular (venous thromboembolism)” and “antiphospholipid antibody (aPL) testing with solid phase-based assay” [5]”

 

 

5. The diagnosis of APS technically fulfils the new criteria, but the aCL antibody levels over time are borderline. Was the patient tested for other thrombophilia conditions, like Protein C or S deficiency, Factor V Leiden, antithrombin III deficiency, Type 2B vWD, etc? Was any genetic testing for mutations performed?

 

 [Response]

Thank you very much for your important comments.

We have added text and Table to Case presentation (Page 2, Lines 73-74 and Table) as follows:

“A search for causes of thrombophilic predisposition was performed, but no abnormalities were found (Table).”

 

Tests for thrombogenic predisposition are described in the table. However, we have not tested for Type 2B vWD, Factor V Leiden, or even genetic testing for mutations.

 

 

6. It would be helpful to add a chart of the events and therapy similar to Figure 2 for the period between occurrence of the first DVT until vaccination. It is not clear if different medication was subsequent or concomitant. In addition, the age of the first DVT is described first as 6 years then as 5 years.

 

 [Response]

Thank you very much for your important comments.

We have changed to two figures (Figure 1 and Figure 2) for better understanding of the timeline and disease course.

 

The diagnosed age of the patient is 6 years. We have corrected the description (Page 2, Line 77).

 

 

7. Please add references to the sentence (line 86) “At that time, many cases of thrombosis caused SARS-CoV-2 mRNA vaccines had been reported.” I am aware only of rare case reports, which can hardly be characterized as “many cases” and might only be coincidental and not causative.

 

[Response]

Thank you very much for your important comments.

We have changed the case presentation (Page 3, Lines 113-115) as follows:

“At that time, no distinction was made in Japan between adenovirus vector vaccines and mRNA vaccines, and a causal relationship between all SARS-CoV-2 vaccines and thrombosis was suspected.”

 

 

8. Figure 1 is not consistent with the text. For instance, there is information about a PF4 value of 350 ng/ml that is not reflected in the figure.

 

[Response]

Thank you very much for your important comments.

We have changed figure 1 (page 3) and figure 2 (page 4).

And we have revised the case presentation (Page 3, Line 130-133) as follows:

“A second vaccination was administered while taking 81 mg of aspirin to suppress platelet activity, and after one month, the PF4 level decreased to 357 ng/ml and went to study abroad.”

 

 

9. The authors’ conclusion, “The persistence of high PF4 levels from vaccine administration to DVT recurrence suggested APS exacerbation by the vaccine,” is not supported by the data. Did the titer of the antiphospholipid antibodies increase after the vaccination? Did new autoantibodies appear after vaccination? Was the patient tested for anti-PF4 antibodies? Were there other markers of intravascular platelet activation (e.g. sP-selectin or similar) present? If there are plasma samples still available, these tests should be performed.

 

[Response]

Thank you very much for your important comments.

We have added text to Discussion (Page 6, Lines 215-217, Page 6, Lines 219-22) as follows:

“PF4 forms a complex with β2GPⅠ, is recognized by anti-β2GPⅠ antibodies, and induces platelet activation in APS, which may indicate the disease activity of APS [3].”

“And since the patient's aCL-β2GPI was transiently elevated after the second vaccination (Figure 2), it is possible that the patient developed DVT due to a further increase in APS activity after the second vaccination.”

 

In addition, this patient did not undergo ELISA anti-PF4 testing.

We have added text to Case presentation (Page5, Lines 164-166) as follows:

“Because this case of DVT occurred more than 3 months after the second BNT162b2 vaccination, the anti-PF4/heparin IgG ELISA test could not be performed because the likelihood of DVT was considered low.”

 

Furthermore, since there are no plasma samples left at that time, it is not possible to perform the sP-selectin test and other tests.

 

 

10. Please state clearly if the patient was free of any estrogen therapy and was not pregnant.

 

[Response]

Thank you very much for your helpful recommendations.

We have added the following sentence (Page 4, Line 122-23):

“The patient was free of any estrogen therapy and was not pregnant.”

 

 

11. The reference 3 for the statement (L147-148) “DVT is common in patients with trauma who have elevated PF4 and βTG levels, and elevated PF4 levels are correlated with an increased risk of thrombosis [3]” is misleading. The paper cited refers to PF4 and betaTBG mRNA microparticles as a biomarker for DVT, but the plasma levels of PF4 or betaTBG are not included in this paper.

 

[Response]

Thank you very much for your helpful recommendations.

I agree with your comments. This text was deemed inappropriate and has been removed.

 

 

12. The authors seem to follow PF4 levels in many patients. Can they provide information if they noticed a similar increase of PF4 levels in other patients and if they established the cause or circumstances? Did they notice other cases of increased PF4 levels after Covid-19 vaccination?

 

[Response]

Thank you very much for your important comments.

Since my experience with this case, I have measured PF4 in other pediatric patients infected with SARS-CoV-2 virus, but PF4 was not elevated in patients without autoimmune disease or thrombophilic predisposition.

Nor have I encountered any pediatric patients with elevated PF4 after BNT162b vaccination. None of these children had autoimmune disease or thrombophilic predisposition.

I thought that checking PF4 before and after SARS-CoV-2 vaccination, limited to APS patients with a history of DVT, could possibly predict and prevent the development of DVT.

In patients with a similar thrombophilic predisposition as in the present case, if PF4 was elevated, it would be advisable to provide information to alert the patient to the possibility of developing DVT.

13. There is a clear difference between VITT after adenoviral vaccines and TTS after mRNA vaccination. The text implies that VITT is common after mRNA Covid -19 vaccination. This is not true, so please revise this part of the manuscript.

 

[Response]

I agree with your comments. This text was deemed inappropriate and has been removed.

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The authors did not address my main concern about the causative relationship between vaccination, high PF4 levels, APS and exacerbation of DVT. Their conclusions or hypothetical and highly speculative. The authors have no evidence to their main claim that PF4 levels can predict thrombosis.

Comments on the Quality of English Language

English is fine.

Author Response

The authors did not address my main concern about the causative relationship between vaccination, high PF4 levels, APS and exacerbation of DVT. Their conclusions or hypothetical and highly speculative. The authors have no evidence to their main claim that PF4 levels can predict thrombosis.

Response:

Thank you very much for your important comments.

 

We have added text to Discussion (Page6, Lines 191-193, 196-197) as follows:

“However, it has recently been reported that mRNA vaccines also induce transient platelet activity, and further mechanisms will be elucidated in the future [11].”

“The difference between DVT in this case and VITT is speculated to be related to APS.”

 

We have changed text within the Discussion (Page 7, Lines 263-265) as follows:

“However, it should be emphasized that the patient continued to exhibit PF4 levels that were 20–30 times higher than the reference value after vaccination and subsequently developed DVT.”

 

We have changed text within the Conclusion (Page 7, Lines 267-269) as follows:

“In conclusion, it is certain that SARS-CoV-2 vaccination triggers platelet and coagulation activation, so careful follow-up with platelet and coagulation activation markers such as PF4,  βTG, D-dimer and TAT is important.”

 

We have added literature to the References (Page8, Lines 317-319) as follows:

“11. Malika, A.; Kathryn, E. S.; Ai-Ris, Y. C.; Joseph, P. N.; James, V. M.; Steven, E. M.; Dan H. B. Activation of co-agulation and proinflammatory pathways in thrombosis with thrombocytopenia syndrome and following COVID-19 vaccination. Nat commun 2023, 14, 6703. DOI:10.1038/s41467-023-42559-x.”

 

Author Response File: Author Response.docx

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

The message in the paper is considerably improved, But I still think the conclusion is too strong and not valid. I recommend to tone it down, e.g.: In conclusion, SARS-CoV-2 vaccination triggers platelet and coagulation activation in some predisposed patients, so careful follow-up with platelet and coagulation activation markers such as PF4, βTG, D-dimer and TAT is important.

Author Response

Comments:

The message in the paper is considerably improved, But I still think the conclusion is too strong and not valid. I recommend to tone it down, e.g.: In conclusion, SARS-CoV-2 vaccination triggers platelet and coagulation activation in some predisposed patients, so careful follow-up with platelet and coagulation activation markers such as PF4, βTG, D-dimer and TAT is important.

Response:

Thank you very much for your important comments.

We have replaced the sentence (Page 7, Line 267-269) as follows:

“In conclusion, SARS-CoV-2 vaccination triggers platelet and coagulation activation in some predisposed patients, so careful follow-up with platelet and coagulation activation markers such as PF4, βTG, D-dimer and TAT is important.”

Author Response File: Author Response.docx