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K. Althaus et al.
Supplementary Figure S4). In contrast, the platelet popula- tion was almost non-affected after incubation with sera from vaccinated controls (Online Supplementary Figure S5A). Interestingly, the generation of procoagulant platelets was reduced by 0.2 IU/mL LMWH (FI CD62p/PS positive PLT: 13.32±11.50, P=0.016, Figure 6B) and com- pletely inhibited by high concentration of UFH in VITT patients (FI CD62p/PS positive PLT: 1.92±0.96, P=0.008, Figure 6B, Online Supplementary Figure S4). These reactions were also inhibited by the FcγRIIA blocking with mAb IV.3 as well as by high concentrations of IgG (FI CD62p/PS positive PLT: 1.04±0.22, P=0.031 and FI CD62p/PS positive PLT: 7.88±5.56, P= 0.031, respectively, Figure 6B). No significant increase of procoagulant platelets was also observed in presence of PF4 (FI CD62p/PS positive PLT: 37.07±23.73, P=0.078) and in the presence of Spike-RBD alone (FI CD62p/PS positive PLT: 22.02±17.09, P=0.195). While an increased generation of procoagulant platelets was observed after the incubation of sera from severe COVID-19 patients, no significant change was observed when sera from vaccinated individ- uals with anti-PF4 antibodies were tested (Online Supplementary Figure S5A and B).
In order to identify the target antigen of the platelet acti- vating antibodies, the HIPA and FC analyses were repeat- ed at different titrations of sera from VITT patients. Interestingly, diluted sera (from 1:64) were able to activate platelets and induce a procoagulant phenotype only in the presence of PF4 (Figure 7A and B, respectively).
Discussion
The increasing number of reports on rare thrombotic events after SARS-CoV-2 vaccination draw public atten- tion and led to concerns regarding the safety of this vac- cine due to the uncertainty of the origin of these undesired reactions.7-9 In order to understand the pathophysiology of this phenomenon, the so-called VITT, we analyzed sera from eight patients. Our mostly young, generally fit cohort of patients, presented acutely with atypical throm- bosis, primarily, but not exclusively involving the cerebral venous sinuses, an extremely rare manifestation of throm- bosis in the general population. All cases developed symp- toms within 6-20 days after the ChAdOx1 nCoV-19 vac- cination showing a temporal relationship between vacci- nation and symptoms. The main findings in these cases were thrombocytopenia, high D-dimer, low fibrinogen, and high-titer IgG antibodies against PF4 that can induce procoagulant platelet phenotype.
After intensive laboratory investigations of the VITT cases, we were able to identify the serological profile of the pathological antibodies. In a small cohort of vaccinat- ed volunteers, approximately 10% of the individuals developed IgG antibodies against PF4/polyanion complex- es within 14 days after the first vaccination; none of them had been exposed to heparin in the past 100 days. We observed that IgG binding to PF4 in these sera as well as in VITT sera can be inhibited by heparin but also by increasing the concentration of Spike-RBD. These data may suggest that these antibodies are specific for confor- mational changes in PF4 that might be induced by nega- tively charged structures. Of note, no significant IgG bind- ing to platelets was observed in the presence of the vac- cine ChAdOx1 nCoV-19. Accordingly, it is very unlikely
that the vector (pCDNA4) may be responsible for the high PF4-seroconversion rate in vaccinated individuals. Comparable data were reported by Greinacher et al.7 and Schultz et al.9 in two very recent reports that appeared while our manuscript was in preparation. In addition to their observations, we were also able to demonstrate that sera from VITT patients directly induce procoagulant platelets, suggesting a possible mechanism for thrombotic events seen in patients with VITT. This is further corrob- orated by the pathological studies in two of our patients. Despite the distinct immediate causes of death in these two fatal cases, namely fatal cerebral sinus thrombosis and intracerebral hemorrhage, the two autopsy reports showed striking similarities. In addition to arterial, arteri- olar and venous thrombosis in various organs and pul- monary thromboembolism, both cases showed a striking occlusion of multiple glomeruli and afferent arterioles by hyaline thrombi composed of fibrin and platelets, but lacking erythrocytes. The kidney morphology bears resemblance to thrombotic microangiopathy, but we failed to identify erythrocyte fragmentation, a key feature of thrombotic microangiopathy.10 Both patients, however, had normal kidney function (highest creatinine level 0.5 mg/dL in case #2 and 0.8 mg/dL in case#3) until briefly before death, indicating rapid pre-terminal development of glomerular microthrombosis. White thrombi have been associated with antibody-mediated platelet activation.10,11
Our data indicate that IgG antibodies against PF4 increase the generation of procoagulant platelets in VITT. However, we cannot exclude other co-factor(s) that could also induce thromboembolic complications in vivo. We report on VITT after ChAdOx1 nCoV-19, which is the only SARS-CoV-2 vaccine that includes a simian aden- ovirus. Disturbances of platelets have been described in association with the intravenous administration of aden- ovirus gene therapy vectors although it is unclear how that might relate to isolated thrombocytopenia as an adverse event of the vaccine.8
Finally, the observed clinical and laboratory features of the VITT are exceptional and rare. Therefore, the value of the COVID-19 vaccination to provide critical protection should be considered higher compared to the significant health risk of COVID-19. With the better recognition of this rare complication and the availability of efficient ther- apies, the risk-benefit ratio of ChAdOx1 nCoV-19 might be further reconsidered.
Conclusion
Although the incidence of VITT after ChAdOx1 nCoV- 19 vaccination is very low, the mortality rate is high (37.5% in our case series). Since a global vaccination cam- paign is underway and large numbers of people will be vaccinated, an increase in the number of people with this side effect is to be expected, highlighting the importance of a better understanding of the pathophysiology of VITT. In this study, we present immunological and pathological findings in patients with VITT. Furthermore, we show the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.
Disclosures
No conflicts of interest to disclose.
Contributions
KA, PM, UJS, FF, and TB designed the study; JS, MG, MP,
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