A 56-year-old white man with a history of essential hypertension controlled with atenolol received the first ChAdOx1 nCov-19 vaccine in early May 2021. Four days after vaccination, he developed fever, malaise, and persistent headache. On the fifth day following vaccination, he presented with nausea, vomiting, fall from his height, generalized skin rash on the lower limbs, and ecchymosis. He was promptly admitted. His platelet count was 17,000/mm3 (150,000-450,000/mm3), D-dimer 41,000 ng/ml (<500 ng/ml), and fibrinogen 121 mg/dl (200-400 mg/dl). Peripheral smear showed no platelet clumps or schistocytes. Brain computed tomography (CT) examination identified right frontal heterogeneous intraparenchymal hematoma, measuring approximately 6.4 cm x 5.2 cm x 4.8 cm with a thin hypodense halo, causing mass effect with a local reduction in the amplitude of the sulci, compression over the right lateral ventricle resulting in contralateral deviation of midline structures by 0.7 cm, in addition to areas of bilateral frontoparietal subarachnoid hemorrhage. He also presented with a massive ventricular hemorrhage filling in the right lateral ventricle, the posterior horn of the left lateral ventricle, and the fourth ventricle, and bleeding in the right Sylvian cistern and perimesencephalic cistern. There was no evidence of aneurysmal dilatation. The CT angiogram the following day identified thrombosis in the superior sagittal sinus. Also, a lower extremities Doppler ultrasound showed right arterial and venous thrombosis. He underwent urgent neurosurgery for hematoma drainage and decompressive craniectomy. A few hours after the procedure, he developed new bleeding and bilateral cerebral edema and received plasma, cryoprecipitate, fibrinogen concentrate, platelet transfusions, and 70 g (1 g/kg) intravenous immunoglobulin. Despite all measures, the patient died with refractory intracranial hypertension on day 13 after vaccination.
Patient's relatives and healthy unvaccinated controls provided written informed consent approved by the local Ethics Committee for clinical and laboratory investigations (CAAE #68118417.6.0000.5248 and #48532621.8.0000.5262, respectively). SARS-CoV-2 RT-PCR of the nasopharyngeal swab and serology to dengue, Chikungunya, Zika, HIV, hepatitis B and C, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), toxoplasmosis, and rubella were negative. Relatives denied any past COVID-19 infection or heparin exposure. There was no personal or family history of thrombosis or miscarriages.
IgG anti-PF4 antibodies were detected with a 3.33 optical density (reference <= 0.4). A flow cytometry-based assay to detect platelet-activating antibodies was performed according to Handtke et al. (Figure 1A). When added to healthy donor platelets, patient plasma elicited increased expression of CD62p to a greater extent than plasma from healthy heterologous donors. However, in the presence of high concentrations of heparin, which can destabilize PF4/adenovector aggregates due to its higher affinity to PF4, platelet activation levels were reduced to control levels, confirming the presence of platelet-activating immunocomplexes in the patient's plasma. Elevated plasmatic levels of CD62p, released by activated platelets and endothelial cells, and of tissue factor (TF, coagulation factor III), the primary activator of the extrinsic pathway of the coagulation cascade, corroborate the extensive platelet activation and clot formation (Figures 1B,C). Additional laboratory results are characterized in Table 1. The results of other blood tests were unremarkable except for increased alanine aminotransferase, C-reactive protein, IL-1beta, and caspase-1. Antinuclear antibodies, anti-cardiolipin IgG and IgM, lupus anticoagulant, and beta-2 glycoprotein 1 IgG were not detected.
We performed genetic analysis using the Axiom  Human Genotyping SARS-CoV-2 Research Array, which genotypes more than 870,000 single-nucleotide polymorphisms (SNPs) in the human genome. The first strategy consisted of screening mutations in 232 autosomal genes essential for thrombotic syndromes, to inflammatory disorders, and related to type I interferon (IFN) signaling (Table 2). Aiming to select rare variants, the minor allele frequency up to 0.01 in European or African populations was settled as a cutoff. Six thousand four hundred sixty-six related SNPs were present in the array, and 5,953 are described in the 1KGP database. From the selected SNPs, 689 and 845 rare putative variants were found in databases of African and European populations, respectively. Among them, 68 were found in heterozygosity in the patient. From these, seven SNPs have been studied in clinical conditions; four were considered benign; one likely benign; one, rs116667976, in the Factor XI gene (F11) with conflicting interpretations of pathogenicity; and the last one, rs2884737, in the VKORC1 gene, associated with Warfarin drug response. A descriptive analysis of the SNPs is depicted in Table 3.
In addition to the search for rare variants, a second strategy was employed. We performed a screening of common mutations in the European or African populations that were in homozygosity in the patient. We assessed classical hereditary thrombophilia-associated mutations: Factor V Leiden G1691A (rs6025), Factor II G20210A (rs1799963), and methylenetetrahydrofolate reductase (MTHFR), C677T (rs1801133) and A1298C (rs1801131), in addition to distinct mutations on F11 and genes related to type I IFN. The patient was homozygous for the missense MTHFR variant c.665C > T chr1-11856378 G > A p.Ala222Val NM_005957.5 rs1801133, with clinical relevance for methotrexate drug response, with a general population frequency of approximately 0.3. His medical records from January 2021 and 2008 showed normal levels of folic acid and homocysteine, respectively. Also, he was not on vitamin B12 supplementation or had ever had hemolysis. Furthermore, he was homozygous for the variants in FXI rs2036914 and rs4253405; in TLR3 rs6849187 and rs6857595; in IFNW1 rs7852828; in TBK1/RASSF3 rs1245035 and rs1520765; in TICAM1 rs4807643 and rs8102626; and in IFNAR2 rs2252650. Besides rs2036914, classified as benign, none of these variants have been described as associated with clinical diseases (Table 3 in gray).