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COVID-19 Vaccine Thrombosis: Why is PF4 antibody the key evidence for?

COVID-19 Vaccine Thrombosis: Why is PF4 antibody the key evidence for? NEJM: Thrombosis occurred after being vaccinated with coronavirus vaccine. Why is PF4 antibody the key evidence?

Recently, the “New England Journal of Medicine” published three consecutive research papers from Germany, Norway and the United Kingdom, pointing out that the Oxford AstraZeneca COVID-19 vaccine (AZD1222) has the potential to cause thrombosis and thrombocytopenia. [1～3]

Patients with related symptoms have a large number of platelet factor 4 (PF4) antibodies. Researchers suggest that this type of condition is called “vaccine-associated immune thrombotic thrombocytopenia” (VITT).

COVID-19 Vaccine Thrombosis: Why is PF4 antibody the key evidence for?
NEJM screenshot

Another new coronavirus vaccine that also belongs to the adenovirus vector route has also reported the risk of thrombosis.

According to the New England Journal of Medicine on April 16, a 48-year-old woman developed discomfort and abdominal pain 2 weeks after receiving the Johnson & Johnson Covid-19 vaccine (Ad26.COV2.S). The examination found that the platelets were severely reduced and the patient had cerebral venous sinus thrombosis (CVST). ), the PF4/polyanionic polymer antibody was found to be positive in the test. [4]

In these cases, it is not difficult to see that “PF4 antibody” is a word that has been mentioned repeatedly.

What role does this antibody play in the coagulation process? Why PF4 antibody may be the key evidence to help clinical identification of VITT?

What role does PF4 antibody play in the coagulation process?

First, we need to understand another disease: heparin-induced thrombocytopenia (HIT).

Why does the anticoagulant heparin cause blood clots instead?

As the name suggests, this is an autoimmune disease that occurs after the human body uses the anticoagulant heparin.

In HIT patients, the immune system secretes antibodies to platelet factor 4 (PF4). The PF4 protein itself has a unique chemical structure and is not easily recognized by antibodies. Therefore, although there are a large number of antibodies in the body of HIT patients, they can remain disease-free for a long time without exposure to heparin.

Once these people receive heparin treatment again due to medical factors, the PF4 protein will directly generate the opportunity to contact with the antibody under the match of heparin, causing the platelets to be stimulated by the antibody to produce spontaneous agglutination.

In other words, the real cause of the disease is the autoantibody (HIT antibody) against the PF4-heparin complex.

The antibody can activate platelets and cause thrombosis. After platelets are activated, more PF4 will be released, forming a vicious circle. At the same time, because platelets in the blood circulatory system have been consumed in large quantities, the number of platelets has been severely reduced, and patients will experience catastrophic arterial and venous thrombosis, which may be life-threatening and the mortality rate can reach as high as 20%.

COVID-19 Vaccine Thrombosis: Why is PF4 antibody the key evidence for?
In VITT patients, platelets aggregate disorderly in the blood vessels, leading to thrombosis and platelet depletion
(Photo source: Paul Erich Institute)

The researchers who proposed the concept of vaccine-related immune thrombotic thrombocytopenia (VITT) speculated that the occurrence of VITT is similar to the mechanism of HIT, and the process involves the participation of PF4 antibodies.

But the difference between the two is that in the two reports in Germany and Norway, patients have no history of heparin use. [1, 2]

In HIT, the participation of heparin is needed to see platelet activation. In VITT, no additional assistance from heparin is needed. Only the PF4 antibody in the patient’s serum can activate platelets (researchers speculate that the vaccine plays the role of heparin). [3]

Then the next question is that platelet factor 4 (PF4) is clearly a protein secreted by the body itself, why do people produce antibodies to their own protein?

This has been a mystery for a long time, and a study has suggested a possible direction.

Why are PF4 antibodies produced?

Previously, Dr. Cai Zheng from the University of Pennsylvania and others explained the mystery of thrombosis caused by HIT antibody through the study of the crystal structure of the complex of antibody and PF4. [7, 8] (Note: The author of this article is the corresponding author of the [7] research)

COVID-19 Vaccine Thrombosis: Why is PF4 antibody the key evidence for?
Antibodies screenshots

Previous studies in this field have shown that PF4 can bind to heparin, and a PF4 monoclonal antibody called KKO can induce HIT.

The crystal structure obtained by Cai Zheng shows that KKO actually binds to the tetrameric PF4 structure, and the core of this structure is a heparin-like polysaccharide molecule.

If we use candied haws as a metaphor, the polysaccharide molecule of heparin is a stick, and the hawthorn fruit that is worn on the stick is the tetramer PF4.

COVID-19 Vaccine Thrombosis: Why is PF4 antibody the key evidence for?
KKO binds to tetramer PF4 and heparin-like polysaccharide molecules

There is originally heparin and PF4 in the human body, but under normal circumstances, the concentration is not high and such “candied haws” will not be formed. It can only be formed after heparin is injected.

Because of its special structure, it may cause immune cells to think that it is an unnatural structure and induce the production of antibodies.

Antibodies like KKO can further stabilize the “candied haws” structure, and because a large number of antibodies are attached to the candied haws, a huge immune complex is formed, which is also the key to the reduction of platelets.

It is worth noting that the human body’s antibodies to PF4 include not only KKO, which recognizes tetramers, but also another antibody that only recognizes monomers. One of the representatives is RTO.

The binding site of RTO on PF4 is not exactly the same as that of KKO. The most important thing is that PF4 is not easy to form tetramers after binding to RTO, and it is not easy to form candied fruit to cause thrombosis.

Different combinations of KKO and RTO

Recognizing that PF4 has two types of antibodies with completely different functions (let’s call it one good and one bad), one can understand why “the presence of PF4 antibodies” is not the only condition for diagnosing HIT. If only PF4 antibodies are screened, the vast majority of patients will have false positives, and only a few will have HIT.

To diagnose HIT, in addition to screening for PF4 antibodies, it is also necessary to see whether the patient has used heparin and whether there is a severe reduction in platelets.

Since PF4 antibodies are “good” or “bad”, can “good” antibodies such as RTO be used to prevent HIT from occurring?

The results of the current in vitro experiments show that it is indeed possible [8], but whether it can be used for treatment requires clinical trials of humanized RTO antibodies to verify.

Going back to this VITT related to the COVID-19 vaccine: the patient has not used heparin, which means that the candied fruit does not need the stick of heparin to be strung together to induce immune cells to produce “bad” antibodies. Researchers speculate on what ingredients are “playing the role of heparin” in the vaccine?

In view of the fact that the patient has problems after receiving a dose of the vaccine, it means that the human body has been exposed to the antigen before, and the immune memory cells already exist.

Both are adenovirus vector vaccines (AZD1222 uses an adenovirus carried by chimpanzees, and Ad26.COV2.S is a human adenovirus with serotype 26). Does the adenovirus provide some kind of candied fruit? stick? Does the origin of HIT and VITT actually originate from some kind of virus infection?

There is currently no answer to this question, and more research is needed to answer it.

Why are VITT female patients mostly?

There is also the fact that many people are concerned about the fact that of the 39 VITT cases related to AstraZeneca’s AZD1222 vaccine, 27 are women, accounting for 69%. The 6 cases related to Johnson & Johnson Ad26.COV2.S vaccine are all women.

Why is there such a difference?

Judging from the current situation, VITT mainly occurs in relatively young people. The 6 women associated with the Ad26.COV2.S vaccine are 18 to 48 years old, belonging to the childbearing age. Therefore, some people have previously suspected that patients taking birth control pills may cause thrombosis. However, this factor was quickly ruled out, and follow-up investigation found that none of these cases had a history of taking birth control pills.

At present, there is no very accurate explanation for this phenomenon. I would like to make a few personal guesses here.

The mechanism of thrombus formation is related to the cross-linking and polymerization of antibodies to antigens. HIT and VITT are all immune diseases. In similar autoimmune diseases (such as rheumatoid arthritis, etc.), women are also at higher risk than men, and the ratio of men to women in patients is 1:3. Autoimmune thyroid diseases, multiple sclerosis, systemic lupus erythematosus, Sjogren’s syndrome, etc. all have the phenomenon of “preferring women to men”. [9]

Male hormones (testosterone) levels are higher, and testosterone is known to have immunosuppressive functions. Among men vaccinated for seasonal flu, too high testosterone is associated with decreased antibody response. [10] In addition, in terms of age, young people have relatively stronger immune functions.

Based on the above reasons, VITT patients are currently mainly “relatively young women”. However, this is only a superficial guess, and it does not explain why most young women have no problems after being vaccinated with these two vaccines.

Implications for treatment options

Obviously, the PF4 antibody provides us with a clue to solve the case and cannot explain the answers to all questions. However, depending on the clues provided by PF4 antibodies, treatment options should be carefully selected for HIT and VITT patients.

Thrombosis in HIT patients cannot be treated with heparin. Heparin can aggravate HIT, especially if normal non-low molecular weight heparin is used.

For VITT, 4 out of 11 cases in Germany used heparin to treat thrombosis. As a result, 2 people died and 2 people were recovering. Another person used low-molecular-weight heparin, and platelets increased. Later, due to the detection of PF4 antibodies, the patient stopped using small-molecule heparin and switched to oral non-heparin anticoagulants. He is currently recovering. [1]

Among the five cases in Norway, one person was treated with heparin and unfortunately died; the other four were treated with low-dose or reduced-dose low-molecular-weight heparin, two died and two fully recovered. [2]

Therefore, although it is not clear how heparin affects VITT, some researchers suggest that non-heparin anticoagulants should be used in the treatment. [11]

Reference 11 Screenshot

In addition, from the treatment results of existing VITT cases, intravenous immunoglobulin (IVIG) and high-dose glucocorticoid use can improve platelet counts within a few days, thereby reducing the risk of bleeding, especially when using anticoagulation During treatment.

IVIG can block the activation of Fc receptors and reduce the further activation of platelets by immune complexes; glucocorticoids can reduce excessive immune responses. These programs are reasonable for an immune disease like VITT.

Of course, this is not the most ideal treatment plan for VITT. With the deepening of research, there may be better treatment plans.

According to statistics from the World Health Organization (WHO) and the European Medicines Agency (EMA), more than 20 million people worldwide have been vaccinated with AZD1222 vaccine, and a total of 469 cases of thrombosis have been reported.

Therefore, EMA believes that these VITT adverse events are rare events, and the benefits of vaccinating the COVID-19 vaccine to prevent infection are still far greater than the risk of adverse reactions such as thrombosis.

Reference:

[1] A. Greinacher, T. Thiele, T.E. Warkentin, K. Weisser, P.A. Kyrle, S. Eichinger, Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination, New England Journal of Medicine, (2021).
[2] N.H. Schultz, I.H. Sørvoll, A.E. Michelsen, L.A. Munthe, F. Lund-Johansen, M.T. Ahlen, M. Wiedmann, A.-H. Aamodt, T.H. Skattør, G.E. Tjønnfjord, P.A. Holme, Thrombosis and Thrombocytopenia after ChAdOx1 nCoV-19 Vaccination, New England Journal of Medicine, (2021).
[3] M. Scully, D. Singh, R. Lown, A. Poles, T. Solomon, M. Levi, D. Goldblatt, P. Kotoucek, W. Thomas, W. Lester, Pathologic Antibodies to Platelet Factor 4 after ChAdOx1 nCoV-19 Vaccination, New England Journal of Medicine, (2021).
[4] K.-L. Muir, A. Kallam, S.A. Koepsell, K. Gundabolu, Thrombotic Thrombocytopenia after Ad26.COV2.S Vaccination, New England Journal of Medicine, (2021).
[5] J. Sadoff, K. Davis, M. Douoguih, Thrombotic Thrombocytopenia after Ad26.COV2.S Vaccination — Response from the Manufacturer, New England Journal of Medicine, (2021).
[6] C.T. Rentsch, J.A. Beckman, L. Tomlinson, W.F. Gellad, C. Alcorn, F. Kidwai-Khan, M. Skanderson, E. Brittain, J.T. King, Y.-L. Ho, S. Eden, S. Kundu, M.F. Lann, R.A. Greevy, P.M. Ho, P.A. Heidenreich, D.A. Jacobson, I.J. Douglas, J.P. Tate, S.J.W. Evans, D. Atkins, A.C. Justice, M.S. Freiberg, Early initiation of prophylactic anticoagulation for prevention of coronavirus disease 2019 mortality in patients admitted to hospital in the United States: cohort study, BMJ, 372 (2021).
[7] Z. Cai, M.I. Greene, Z. Zhu, H. Zhang, Structural Features and PF4 Functions that Occur in Heparin-Induced Thrombocytopenia (HIT) Complicated by COVID-19, Antibodies, 9 (2020) 52.
[8] Z. Cai, S.V. Yarovoi, Z. Zhu, L. Rauova, V. Hayes, T. Lebedeva, Q. Liu, M. Poncz, G. Arepally, D.B. Cines, M.I. Greene, Atomic description of the immune complex involved in heparin-induced thrombocytopenia, Nature communications, 6 (2015) 8277.
[9] R.F. van Vollenhoven, Sex differences in rheumatoid arthritis: more than meets the eye, BMC Medicine, 7 (2009) 12.
[10] A. Goren, F.A. Cadegiani, C.G. Wambier, S. Vano-Galvan, A. Tosti, J. Shapiro, N.A. Mesinkovska, P.M. Ramos, R. Sinclair, O. Lupi, J. Hercogova, J. McCoy, Androgenetic alopecia may be associated with weaker COVID-19 T-cell immune response: An insight into a potential COVID-19 vaccine booster, Medical hypotheses, 146 (2021) 110439-110439.
[11] D.B. Cines, J.B. Bussel, SARS-CoV-2 Vaccine–Induced Immune Thrombotic Thrombocytopenia, New England Journal of Medicine, (2021).