1 Introduction
The World Health Organization declared the coronavirus disease 2019 (COVID-19) pandemic on March 11, 2020 (1). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen causing this infection, has a mortality rate of 2.3% (China) to 7.2% (Italy) and high transmissibility (2, 3). Globally, as of June 15, 2021, there have been 176,156,662 confirmed cases, and among them, 3,815,486 deaths have been reported (4). SARS-CoV-2’s viral characteristics together with globalization of the infections have considerably reduced the possibility of infection containment despite various preventive measures implemented by different governments worldwide. The acceleration of COVID-19 vaccine development has become necessary due to this global state of emergency (5). A few vaccines have been developed and licensed for clinical use in <1 year.
Two such vaccine types were developed using two different technologies: viral vectors, herein adenovirus, and mRNA. Both vaccine types aim to induce an immune response and generation of neutralizing antibodies against the SARS-CoV-2 spike protein. The spike protein, expressed on the virus surface, enables the virus to bind to human target cells and upon entry to reproduce itself. Viral vector vaccines, such as Vaxzevria (AstraZeneca), exploit a weakened version of chimpanzee adenoviruses rendered able to enter and reproduce itself within human cells. The adenovirus codes for the SARS-CoV-2 spike protein in the nucleus, which, upon translation in the cytoplasm and expression, triggers an immune response (6, 7). The COVID-19 mRNA vaccines, such as Comirnaty (Pfizer) and mRNA-1273 (Moderna), contain mRNA molecules encoding spike proteins within human cells (8, 9).
As of June 15, 2021, a total of 2,310,082,345 individuals have been vaccinated (4). However, little is known regarding these experimental vaccines, whose phase IV clinical trial is monitored during the current mass vaccination and will be necessary for identifying, cataloging, and quantifying adverse reactions, since the large sample size would compensate for the reduction in the development and licensing of the vaccine (10).
Different types of vaccine adverse effects have been described, such as anaphylaxis, fever, joint and muscular pain, headache, weakness, nausea, chills, erythema, lymphadenopathies, paresthesia, vomiting, dizziness, arrhythmias, changes in blood pressure, and facial paralysis (11). Thrombosis is one of the most severe and nontypical adverse effects. Some authors have hypothesized an incidence of approximately one in 100,000/1,000,000 individuals who received the adenoviral vector vaccine (12).
Our study aimed to analyze literature reports of thrombosis after COVID-19 vaccine administration to identify patients’ features, potential pathophysiological mechanism, timing of adverse events, and critical issues to prevent further deaths adding to those that are COVID-19 related.
4 Discussion
The current mass vaccination led to the discovery of vaccine-induced thrombotic thrombocytopenia (VITT), an unknown nosological phenomenon secondary to the inoculation of the COVID-19 vaccine (13–15, 18, 21). The pathophysiological mechanism behind this adverse reaction remains unclear. Recent studies have assumed a mechanism that is similar to heparin-induced thrombocytopenia (HIT) (13). The latter represents a known adverse reaction that remains one of the most serious complications in individuals treated with heparin (23). A platelet count decrease of >50% and a procoagulant state with a secondary development of venous or arterial thrombosis represent the main diagnostic criteria from a clinical laboratory point of view (24).
There may be an immune-mediated mechanism at the root of thrombosis, with protagonist antibodies against the PF4–polyanion complex. VITT-associated PF4 antibodies interact with the heparin-binding site. These antibodies are therefore independent from heparin. The immune complexes, formed from the binding of PF4 to antibodies, activate platelets through FcγRIIa receptors, causing thrombocytopenia and thrombosis secondary to the activation of the coagulation pathway. Therefore, in vitro, heparin does not increase the activation of platelets in case of thrombosis with thrombocytopenia syndrome (TTS); heparin could inhibit platelets’ activation secondary to a pharmacological antagonism. The independence from heparin denotes a similarity between TTS and HIT due to heparin-independent antibodies; the main differences consist of the needed previous exposure to heparin in HIT and its greater incidence rate than VITT. The antibodies of HIT heparin independent and TTS have a high affinity for the receptor and are able to bind it in the absence of heparin. Previous studies discovered that HIT heparin-independent antibodies can activate platelets in the presence of adenoviral vector and in a dose-related manner. In fact, HIT is also caused by various mechanisms, which include polymorphism of FcγRIIa receptor (25), monocyte activation, production of tissue factor (26), and generation of procoagulant microparticles (27). These findings suggest that the PF4 antibodies-mediated platelet activation may not be the only cause for thrombosis in VITT (13). In fact, polyanions including nucleic acids and components of the bacterial cell wall, with their negative charge, promote binding to PF4, a positively charged chemokine released by platelets during platelet activation (28). It is plausible that the adenoviral SARS-CoV-2-specific proteins could trigger the immunopathological mechanism in VITT; otherwise, other DNA-based vaccines may also cause thrombosis. Antibody cross-reactivity through molecular mimicry between spike proteins and PF4 epitopes has been hypothesized, albeit not yet proven (29). Regarding nucleic acids, binding with PF4 occurs when their plasma concentration exceeds the normal upper limit of 200 ng/ml (30). The viral DNA has a negative charge and can bind to PF4, acting as an adjuvant because of its phosphate groups that can stimulate Toll-like receptor (TLR)-9 on plasmacytoid dendritic cells with the subsequent production of interferon (IFN) alpha (31). RNA is also negatively charged; therefore, it has not been entirely excluded as a cause of cascade of pathological events. The local concentration of nucleic acids increases due to their release by specific cell structures under particular conditions, such as the breakdown of viral and bacterial pathogens, massive tissue damage, and increased apoptosis (32–34). Several authors have reported a correlation between this pathological event and bacteremia, even when it is subclinical, as in the case of periodontitis (33). Other authors have also recently highlighted the viral capacity to trigger this pathological process (35, 36). Moreover, major surgeries, such as knee replacement, may constitute another risk factor (37–40). New experimental studies on vaccine-related thrombosis have recently been performed, suggesting a new possible mechanism (41). They assumed that thrombosis is related to a soluble adenoviral protein spike variant, originating from splicing events, which cause important endothelial inflammatory events, binding to endothelial cells expressing ACE2. This mechanism may also be related to severe cases of SARS-CoV-2 and pseudovirus infections. This would explain the greater correlation of thrombosis with the adenovirus-based rather than with mRNA-based vaccines.
It is also possible that there is a common physiopathological mechanism for COVID-19 infection and VITT or HIT. Some researchers hypothesized an important role of the innate immune system in causing thrombotic events in COVID-19 patients and in cases of VITT or HIT (42).
Neutrophils, which are cells of the innate immune system, are able to capture microorganisms through neutrophil extracellular traps (NETs), which are essential structures for phagocytosis. Dysregulation of this mechanism is responsible for the procoagulant state and the subsequent alveolar damage and coagulopathy of COVID-19 patients (43).
Inflammation and infection are known to stimulate neutrophil recruitment (44). Excessive activation of neutrophils in COVID-19 patients could therefore trigger the coagulopathy secondary to the fibrin deposition during phagocytosis (45). NETs’ components, such as cell DNA, histones, and tissue factor (III) can activate the coagulation. The histones stimulate activation of platelets and endothelium interacting with TLR2 and TLR4 (46–48); on the other hand, platelets induce NETs’ production through P-selectin (49). The NETs expulsion facilitates adhesion and further activation of platelets leading to a self-perpetuating process. Finally, the deposition of fibrin, secondary to NETs, traps bloodstream cells, such as platelets and red blood cells, providing a basis for blood clot formation (49, 50).
In case of VITT, the certain inflammatory vaccine adjuvants and delivery systems could trigger immune system cells’ recruitment, especially neutrophils, through the activation of NPL3 inflammasome and enhance NET production (42, 51, 52). NETs’ components by binding to PF-4 determine the production of antibodies against PF-4 polyanionic complexes.
Concerning the immunological mechanism, it has been hypothesized as either an innate or humoral-mediated response, through the activation of B and T cells, could take part in the pathophysiological mechanism of thrombosis (53–55).
It is assumed that genetic predisposition is based on HIT. Some authors have shown a higher frequency of T-cell death-associated gene 8 (TDAG8) regarding genetic predisposition (56).
Other researchers identified individuals with the FcγRIIA-131RR genotype as a susceptible population, as they could be more efficiently able to activate a procoagulation response by the activation of platelets and monocytes (57, 58).
Therefore, it has been further suggested that monocytes precede the activation of platelets: FcγRIIA receptor triggers a downstream response involving tyrosine kinase-dependent pathway, with a subsequent release of thrombin and tissue factor, which are both coagulation promoters (59).
To date, a correlation of VITT with a specific medical history remains unknown.
Interestingly, in our study, the overall patient samples consisted of men in the articles on the Pfizer and Moderna vaccines, while the patient samples were predominantly female in the articles on the AstraZeneca vaccine. These findings should also be further analyzed to evaluate their significance in larger sample sizes.
The diagnostic criteria of VITT are the following: time of onset ranging from 4 to 30 days after the AstraZeneca or J&J administration, diagnosis of thrombosis, finding of thrombocytopenia, and PF4 HIT ELISA positivity.
The evaluation of the increase in anti-PF4 antibodies in VITT is performed differently than that of HIT because of the absence of hospitalization and monitoring of laboratory parameters after vaccination. Consequently, the increase in antibodies in VITT can be evaluated indirectly by analyzing the time relationship between vaccination and the onset of symptoms. This temporal range is similar to that of HIT in our study, which is in accordance with the literature (23).
The anti-PF4/heparin antibody titers, in fact, has a characteristic temporal pattern: they appear 4–5 to 10–12 days after heparin administration (60) and disappear within 100 days (61). We noticed that symptoms appeared on the 8th to 10th day after vaccination. We can conclude that during the first 15 days after the vaccination, the vaccinees are at high risk of symptom development, and this period may represent the ideal temporal range in thrombosis prevention. Laboratory data monitoring should therefore preferentially be performed in this temporal range.
Based on our study, the most affected thrombosis site was the cerebral venous system, and this is confirmed by the observations in the literature (23), which also points out frequent involvement of the portal circulation. In fact, the portal circulation and the cerebral venous circulation drain the intestines and nasal sinus, respectively, both sites with a high bacterial presence; it facilitates the passage of bacteria, viral products, or toxins in the veins. The interaction between these elements with PF4 could cause an abnormal immune response with platelet and neutrophil activation, in case of high anti-PF4 antibodies titer (23). According to another recent hypothesis, the major incidence of thrombosis in these venous sites could be related to a non-unidirectional blood flow due to body posture in this vascular district, which does not present a valvular system, with a prolonged exposure to the soluble spike protein and a high risk of binding of this protein to endothelial cells expressing ACE2 (41). These sites are different than those affected in the HIT, including adrenal hemorrhagic necrosis, limb thrombosis, disseminated intravascular coagulation, skin necrosis, deep vein thrombosis, left ventricle thrombosis, and endograft thrombosis (62–69).
Cerebral venous sinus thrombosis (CVST) represents a rare pathological disease occurring secondary to blood clots, which obstruct the blood flow of cerebral vessels or dural sinuses (70).
Women of child-bearing age are the most affected subjects (71, 72). The major risk factors are anticoagulation therapy, brain infections, head trauma, pregnancy, and contraception (73, 74). COVID-19 patients presented relatively low incidence of this complication. Recently, some individuals, especially women aged between 18 and 60, developed CSVT after vaccinations with the AstraZeneca and Johnson and Johnson (Ad26.COV2.S) vaccines. The occurrence of these complications temporarily prohibited administration of the J&J vaccine, in the USA and Germany, and of AstraZeneca vaccine in many European countries (75).
The mRNA vaccines, on the other hand, are not associated with this complication (76). CSVT incidence rate hovers around three to four cases per million before COVID-19 vaccinations (77). The incidence rate of CSVT is 1 in 100,000 after AstraZeneca and 1 in 1,000,000 after J&J vaccinations (78).
Clinical presentation includes headache, generalized or localized, in association with hemiparesis and aphasia. Seizures and encephalopathy appear in a minority of such cases, and intracranial hypertension is responsible for their onset.
Secondary symptoms therefore include dyspnea, limbs’ weakness, petechiae, and lethargy (76).
Randomized controlled trials recommended anticoagulation therapy, except for heparin, in order to avoid the extent of the hemorrhage (77, 78). In fact, heparin and heparin-containing products are not recommended due to the risk of progression of thrombosis and the correlation with HIT for the above-mentioned reasons (13).
Some authors propose the administration of plasma exchange, in order to increase the fibrinogen level, or even the use of Bruton tyrosine kinase (Btk) inhibitors. Btk inhibitors could block downstream pathway of FcγRIIA receptor and prevent monocytes and platelets’ activation and NETs’ formation (79).
Finally, the outcome of VITT depends on the extent of venous, arterial, or microcirculatory thrombotic complications (80, 81). We highlighted a negative outcome in all studies reporting these data, except for the largest study with a prevalent positive outcome.
Specific guidelines could be created on the basis of the event’s frequency and the eventual risk of revealing patient identity, similar to those for HIT, recommending close monitoring of at risk patients, every 2–3 days, especially during the above-mentioned time interval of the first 15 days after vaccination (82, 83).
Further studies are needed to better identify VITT pathophysiological mechanisms, the importance of genetic, demographic, or clinical predisposition, the characteristics of high-risk patients, the correlation of thrombosis with the different vaccine types, and the statistical significance of the findings.
4.1 Study Limitations
The small sample size and heterogeneous data collection allowed us to perform an exploratory and descriptive analysis and hence not testing the statistical significance of the findings.