key: cord-0844988-3kduhor3
authors: Kashir, Junaid; Ambia, Ayesha Rahman; Shafqat, Areez; Sajid, Muhammad Raihan; AlKattan, Khaled; Yaqinuddin, Ahmed
title: Scientific premise for the involvement of neutrophil extracellular traps (NETs) in vaccine‐induced thrombotic thrombocytopenia (VITT)
date: 2021-09-01
journal: J Leukoc Biol
DOI: 10.1002/jlb.5covr0621-320rr
sha: 5efcefe8ee7100a882d2b986f9c1e1249608bd36
doc_id: 844988
cord_uid: 3kduhor3

Following on from the devastating spread of COVID‐19, a major global priority has been the production, procurement, and distribution of effective vaccines to ensure that the global pandemic reaches an end. However, concerns were raised about worrying side effects, particularly the occurrence of thrombosis and thrombocytopenia after administration of the Oxford/AstraZeneca and Johnson & Johnson's Janssen COVID‐19 vaccine, in a phenomenon being termed vaccine‐induced thrombotic thrombocytopenia (VITT). Similar to heparin‐induced thrombocytopenia (HIT), this condition has been associated with the development of anti‐platelet factor 4 antibodies, purportedly leading to neutrophil‐platelet aggregate formation. Although thrombosis has also been a common association with COVID‐19, the precise molecular mechanisms governing its occurrence are yet to be established. Recently, increasing evidence highlights the NLRP3 (NOD‐like, leucine‐rich repeat domains, and pyrin domain‐containing protein) inflammasome complex along with IL‐1β and effete neutrophils producing neutrophil extracellular traps (NETs) through NETosis. Herein, we propose and discuss that perhaps the incidence of VITT may be due to inflammatory reactions mediated via IL‐1β/NLRP3 inflammasome activation and consequent overproduction of NETs, where similar autoimmune mechanisms are observed in HIT. We also discuss avenues by which such modalities could be treated to prevent the occurrence of adverse events and ensure vaccine rollouts remain safe and on target to end the current pandemic.

transverse myelitis, Guillain-Barre syndrome, immune thrombocytopenia, disseminated intravascular coagulation (DIC), anaphylaxis, encephalomyelitis, narcolepsy, pericarditis, cerebral venous sinus thrombosis (CVST), splanchnic vein thrombosis (SVT), and appendicitis. [5] [6] [7] There is an immediate need to elucidate whether these AESIs and vaccines share a causal relationship to further optimize vaccine protocols, provide therapeutic strategies, and reduce public fear and hesitancy over the vaccines. 8 It should, however, be emphasized beforehand that the vaccines are largely safe, as AESIs are exceptional findings, and the benefits of receiving the vaccine far outweigh any potential risks.

Reports of thrombosis in atypical locations, particularly CVST and SVT, with concomitant thrombocytopenia following immunization with the adenoviral ChAdOx1 nCoV-19 AstraZeneca and Ad26.COV2.S Janssen vaccines resulted in these vaccines being temporarily withdrawn in Europe and the United States, respectively. Upon further testing and being declared efficacious and safe, these vaccines were re-authorized for use. Elucidating the mechanisms and investigating potential therapeutic strategies have been the subject of intense study ever since. The temporal coincidence of thrombosis and thrombocytopenia following vaccine administration suggested a mechanism resembling that of heparin-induced thrombocytopenia (HIT), with the term vaccine-induced thrombotic thrombocytopenia (VITT) being coined to refer to this clinical entity. This was further strengthened by the demonstration of anti-platelet factor 4 (PF4) antibodies in the sera of patients afflicted with VITT. [9] [10] [11] Recently, neutrophil extracellular traps (NETs) have gained traction as being key in mediating thrombotic events characterizing severe COVID-19 and various auto-immune conditions, including HIT. [12] [13] [14] [15] Accordingly, in this article, we detail the similarities and differences in the pathogenesis of HIT and VITT, discuss how the different components of NETs facilitate thrombosis, elucidate how NET production could occur in the setting of COVID-19 vaccine administration to subsequently mediate thrombosis and thrombocytopenia, and provide questions that should be addressed by future research to scrutinize the validity of this hypothesis. Last, we briefly review validated therapeutic strategies reported thus far and suggest drugs that could prove to be efficacious by inhibiting NETs. Elucidating whether NETs play a major or minor role in VITT should be the focus of future studies as this could have significant therapeutic implications far beyond the treatment of VITT; further, NET inhibitors could represent viable options to mitigate VITT as well as numerous other prothrombotic disorders.

The Oxford/AstraZeneca vaccine gained rapid emergency approval and clearance for use following confirmation of efficiency ( An article reported 11 patients, aged 22-49 years and 9 of whom were female, who experienced thrombotic events beginning 5-16 d postvaccination with the ChAdOx1 vector vaccine. 7 All patients studied developed thrombosis, with the most common site being CVST, and tested positive for anti-PF4 antibodies and in a PF4-enhanced platelet activation test independent of heparin, demonstrating that thrombosis was mediated by activation of platelets via auto-antibodies against PF4. 18 Accordingly, the clinical criteria for the diagnosis of VITT include 1 exposure to the AstraZeneca or Janssen COVID-19 vaccines 4-30 d before presentation, 2 thrombosis, 3 thrombocytopenia, and 4 positive for anti-PF4 antibodies on a standard ELISA. Besides positive anti-PF4 antibodies, other important lab findings reveal an elevated D-dimer, normal or low fibrinogen levels, and DIC leading to a consumption coagulopathy with consequent hemorrhage. 7, 9 Because the pathophysiology seemed to mimic that of HIT, the term VITT was coined to refer to this clinical entity.

Neutrophils are the most abundant type of circulating cell in the body, and are critical components of the innate immune response against infectious and noninfectious disease pathogenesis. 18 Recently, the expulsion of chromatin by neutrophils to form specialized structures known as NETs has gained attention in describing the function of neutrophils in health and disease. 19 These unique, web-like structures, are composed of DNA, histones, and enzymes, which are released from neutrophils as they undergo a specialized type of cell death called "NETosis." 20 Although the primary function of NETs is to trap microbes and associated debris, an uncontrolled proliferation of NETs from neutrophils culminates in alveolar damage, endothelial injury and coagulopathy ( Fig. 1) . Importantly, NETs are now also increasingly associated with central roles in COVID-19 infection and pathogenesis. 20 Although the role of neutrophils in thrombosis is currently poorly understood, neutrophils are among the first cells that are recruited at inflammation or infection sites, 21 and promote coagulation via fibrin deposition to prevent microbial spread. 22 Excessive activation or dysregulation of neutrophils in blood vessels results in coagulopathy, 23 suggesting the potential importance of NETmediated thrombosis regulation. 19 Indeed, NETs form scaffolds, which trap platelets, RBCs, and platelet adhesion molecules such as fibrinogen, vWF, and fibronectin, 24 which may not only provide the structural basis for thrombosis, but also can potentially induce coagulation through its various constituents by activation of platelets. 25 Indeed, NET-specific markers are significantly elevated in COVID-19 F I G U R E 1 Schematic summary indicating the proposed pathophysiology underlying the role of NETosis in thrombosis. damage-associated molecular patterns (DAMPs), platelets, and complement receptor engagement trigger nonlytic neutrophil extracellular trap (NET) formation, allowing NETing neutrophils to retain basic phagocytic and chemotactic functions. Nonlytic NETosis directly activates PAD-4, which converts arginine to citrulline on histones, causing loss of positive charges of histones and disrupting electrostatic attractions between histones and DNA. The result is decondensation of chromatin. Subsequently, chromatin and associated proteins are lost via blebbing of the nuclear envelope, which is resealed afterward. NETs provide a structural basis for thrombosis and promote platelet aggregation and coagulation, manifesting as a pro-thrombogenic state. Additionally, NETs can lead to the production of ANCA, leading to ANCA-associated vasculitis which has been noted in severe cases of COVID-19 patient sera, whereas Interestingly, COVID-19 patient sera triggered NET production in healthy control-derived neutrophils, 13, [26] [27] [28] [29] and COVID-19 neutrophils exhibited significantly higher levels of NETs at baseline. 29 Although NET release during infections is predominantly considered to be physiologically beneficial, perhaps excessive NET production is harmful, resulting in tissue injury and thrombosis. 21, 30 Although NETosis is physiologically beneficial, when excessive, NETs are key in propagating thrombosis and thrombocytopenia characterizing various disorders, as well as being implicated in the pathogenesis of The general immunologic sequence culminating in NETosis involves the initial activation of macrophages secondary to binding of pathogen-associated molecular patterns or damage-associated molecular patterns (DAMPs) to pattern recognition receptors, most commonly NLPR3 monomers, which form an inflammasome complex through oligomerization. 42 The NLPR3 inflammasome promotes secretion of IL-1β, which positively reinforces inflammasome formation and perpetuates neutrophil recruitment and activation. These NETs provably contribute to vaccine immunogenicity and thereby efficacy. This is due to the use of vaccine adjuvants, which bolster host defense against the immunogen the vaccine delivers. The common mechanism of all adjuvants is the recruitment of innate immune cells, such as neutrophils, dendritic cells, and macrophages, which activate B-and T-cells to confer long-term immunity. Adjuvants are generally considered safer than live-attenuated vaccines and, by increasing vaccine efficacy, they lead to dosage sparing, which increases global vaccine supply. Examples of common adjuvants include aluminum salts, saponins, and emulsions, which activate inflammasomes and proinflammatory cytokines, especially IL-1β. 44, 45 The inflammasome targeted by adjuvants is NLRP3, which is induced through NF-κB signaling.

The temporal association of thrombosis and thrombocytopenia fol- 

With the caveat that detailed vaccine component specifications are not current available, multiple hypotheses also suggest that perhaps either specific factors, or even combinations of such factors, present as vaccine components such as adjuvants may play some kind of role. 58 Indeed, Greinacher et al., 59 COX enzymes (induced via the spike proteins of coronaviruses) can catalyze thromboxane A2 (TxA2) production, 71 perhaps underlying the thromboinflammation observed in VITT. 72 Indeed, in mice infected with SARS-CoV, TxA2 production was markedly increased in younger mice compared to middle aged mice, 72 in line with the higher risk for thrombosis in adults aged <60 yr. 7, 72 Intriguingly, platelets from female mice seem significantly more reactive than from male mice, 73 whereas TxA2 generation, TxA2-platelet interaction, and platelet activation also seem increased in women compared to men. 74, 75 All such observations seem consistent with the apparent increased risk of thrombosis in women following the AstraZeneca and J&J vaccines. 72 However, it is important to note that based on the current evidence, specific risk

factors have yet to be confirmed.

Similar to the management approach in the setting of HIT, a high dose of i.v. immunoglobulin (IVIG) of 1 g/kg for 2 d may also be administered. 1, 9, 10, 76 Initiating a high-dose steroid therapy with or without high-dose immunoglobulins has shown to rapidly increase the platelet count in VITT patients in whom IVIG is delayed or platelet count is <50 × 10 9 . 77 Low molecular weight heparin should not be used in these patients as strong reactivity with antibodies could worsen the condition. Warfarin is also contraindicated as it can potentially worsen thrombosis. Fondaparinux, danaproid, lepirudin, and argotraban can be used to treat thrombotic events in these patients. 78, 79 Many avenues can be considered for the treatment of thrombocytopenia associated with thrombosis, all of which can be associated with varying modes of pathogenesis (Table 2 ). Considering the body of evidence supporting the role of NETs in VITT, it is perhaps prudent to suggest prophylaxis of low-dose aspirin in populations susceptible to VITT, which predominantly seems to be females aged 20-50 yr, administered for 2 wk to prevent this potentially dangerous adverse effect. The basis of this recommendation is the prevention of platelet activations and platelet aggregation by aspirin. As discussed earlier, a complex interplay exists between platelets and neutrophils that induces NETosis and exacerbates thrombosis. Therefore, by abrogating platelet activation and aggregation, NETosis can potentially be prevented. Sivelstat, a neutrophil elastase inhibitor, ameliorates liver damage due to portal vein thrombi and intravascular sinusoidal microthrombi in PHTN and sepsis, and probably numerous other disorders characterized by thrombosis. 31 A more comprehensive review of various drugs shown to inhibit NETs can be found here. 80 Further work is required to fully elucidate the therapeutic potential of these drugs in conjunction with unraveling the role of NETs in various prothrombotic disorders including VITT.

We propose that NETs could play major roles in VITT, as earlier research has firmly established NETs as key in precipitating a prothrombotic state characterizing various disorders, including HIT. To this end, we detailed the various components of NETs propagating thrombosis, and how NET formation in the context of ChAdOx1 nCoV-19 and Ad26.COV2.S vaccine administration could perpetuate VITT.

We also highlighted currently unaddressed research questions that, if answered, would not only further our understanding of VITT but also of a plethora of other thrombotic disorders. Determining if NETs play a major or minor role in VITT will significantly impact pharmacologic approaches to treating this disorder as various drugs are known to inhibit NET formation, or in the early detection of susceptibility to NET-VITT. Specific biomarkers for NETs in body fluids include cell free DNA, MPO-DNA, and Cit-H3, which are easily and rapidly detectable using ELISA-based or strip assays. 13, 27, 81 Therapeutic options that could be investigated include recombinant DNase-1 (dornase alfa) in combination with tPA for resolution of thrombi caused by excessive NET production, whereas NET production was also blocked using histone deacteylase inhibitors and IL6

blockers. 13, 20, 27, 82 Amantadine can block viroporins and calcium influx, which is crucial for PAD-4 activation in NET pathogenesis, 13, 83, 84 whereas NET-induced inflammatory tissue damage, coagulopathy, and vasculitis can be controlled by exogenous administration of resolvins. 13, 27, [85] [86] [87] Ankinara is another potential target that potentially disrupts the IL1-β-NET feedback loop. 13, 20 Another alternative, although no changes in survival rates were observed for treatment of ARDS, includes the neutrophil elastase inhibitor Sivelestat could also be a potential target to tackle NET-induced VITT. 13, 88 Finally, considering the inflammatory-like conditions presented by COVID-19, IVIG administration has been examined as a possible route to reduce proinflammatory markers, with numerous studies now showing that deployment of IVIG within specific dosages would reduce mortality and length of hospitalization of severe COVID-19 patients (refer to Yaqinuddin et al. 1 for a more detailed review). Furthermore, a strong correlation was observed between anti-NET IgG and IgM with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer, potentially impairing NET clearance and exacerbating SARS-CoV-2-mediated thromboinflammation. 89 To this degree, perhaps a therapeutic path in treatment of VITT would be high-dose IVIG administration, which was able to reduce antibody-induced platelet activation in serum in three patients exhibiting heterogeneous symptoms of VITT. 90

This work was supported by a COVID-19 project grant (#C20323) awarded by Alfaisal University to M.R.S.

The manuscript was conceived and context led by A.Y. and J.K. with input and support from A.R.A., A.S., M.R.S., and K.A.K. All authors contributed toward writing the manuscript, which was submitted following the approval of all authors.

The authors declare no conflicts of interest.

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