We present two cases of malignant cerebral infarct, systemic venous thrombosis and concomitant thrombocytopenia in two young healthy adult women within 10 days from vaccination against SARS-CoV-2 with ChAdOx1 nCov-19. High serum levels of antibodies to PF4–polyanion complexes were found in both patients.

Some relevant findings from these two cases need considerations.

Arterial thrombosis in VITT seems to be a rare event comparing to venous thrombosis. A population-based cohort study in Norway and Denmark described an increased rate of venous thromboembolic events but not of arterial events among recipients of ChAdOx1 nCoV-19 (ref. 11). Greinacher et al.2 reported 1 patient, out of 11, with aortoiliac thrombosis whereas Scully et al.4 described two patients, out of 23, affected by MCA occlusion. In Shultz’s case series no arterial thrombosis was reported3. More recently, Blauenfeld et al.5 have reported the first case of VITT in Denmark who developed bilateral adrenal hemorrhage and a fatal massive ischemic stroke treated with decompressive craniectomy, 7 days after receiving the ChAdOx1 nCoV-19 vaccination. Three other cases of VITT presenting with ischemic stroke after the administration of the ChAdOx1 nCoV-19 vaccine in United Kingdom have been described6.

VITT appears to be similar to autoimmune HIT2. Range of thrombotic complications in HIT is broad with 15–20% of patients suffering arterial events, and 30–60% developing venous clots (some patients get a combination of venous and arterial thrombosis)12. The endothelium has been identified as a potential target for HIT antibodies. Recently, it has been demonstrated that VWF strings released from Weibel–Palade bodies activated endothelium, are capable of binding PF4 released from activated platelets. The resulting PF4/VWF complexes forming antigen sites are recognized by HIT antibodies with consequent greater accumulation of platelets to the injured arterial endothelium, thrombus formation, and propagation13.

Patient 1 had a right MCA stroke due to occlusion of the M1 segment. Despite two successful endovascular treatments, the patient experienced a repeated occlusion of the same vessel and developed a malignant MCA infarct. We can hypothesize that the second and the third occlusion at the same vascular site might be due to the PF4/VWF complexes recognized by HIT antibodies on the post-thrombectomy injured arterial endothelium. A high level of VWF and VWF:RCo, D-dimer, and coagulation Factor VIII support this hypothesis, suggesting a high prothrombotic state and endothelium dysfunction. Notably, at the time patient 1 was admitted, we were not yet aware of VITT, as the first paper reporting this syndrome was published a few days later. Hence, finding the low platelet count, a platelet transfusion was prescribed before the first thrombectomy, probably exacerbating the hypercoagulable state.

Histology of the first removed thrombus showed a platelet-rich clot with neutrophils, while the second one showed a red-blood-cell-rich clot. Platelet-rich thrombi are formed by VWF, NETs, and fibrin14. Platelet/fibrin thrombi were also found in the experimental model of HIT in veins and arteries of multiple organs15 and HIT is also known for this reason, as “white clot syndrome”16.

We cannot say whether histological differences of the two thrombi account for different pathophysiology. The main difference between clots was their age. Thrombus formation is a highly dynamic process: first layers of red blood cells and fibrin are main components of thrombotic material, and then platelets and white blood cells become prevalent. The second thrombus was retrieved earlier (about 2 h after its formation), so we can assume that it was fresher than the first one17.

Pathogenesis of the severe normochromic normocytic anemia developed by patient 1 was not defined. We ruled out active bleeding through abdomen CT scan, no melena, or hematemesis were evident. Laboratory findings excluded hemolytic anemia. Bone marrow examination was not done due to the critical conditions of the patient and because of the stability of the red blood cells count after transfusion.

We found a high level of Sars-cov-2 IgG serum antibodies suggestive of a previously COVID-19 asymptomatic infection in patient 1. No Sars-CoV-2 IgG serum antibodies were present in serum of patient 2. These data would suggest that anti-spike protein antibodies might not be relevant in the pathogenesis of this syndrome. Interestingly, antibodies to PF4–polyanion complexes were initially negative in serum of patient 1, while a high antibody level was found in serum collected and tested 2 weeks later.

The serologic pattern of these two cases is similar to that of the so-called autoimmune HIT, since serum from our patients strongly activated platelets in the absence of heparin (heparin-independent platelet activation)18. Moreover, platelet secretion was scarcely sensitive to inhibition with high-dose heparin, as observed in two patients described by Schultz et al.3 and in one described by Greinacher et al.2. A disseminated intravascular coagulation was not observed in our patients.

Both the patients had extensive arterial thrombosis of the lung and venous thrombosis of the splanchnic district. A hypercoagulable state was observed in both the patients with a high level of VWF and VWF:RCo, D-dimer, and coagulation Factor VIII. Factor XIII was markedly decreased in patient 1, probably due to excessive consumption within thrombi19.

Treatment of this syndrome is challenging. Administration of high-dose IVIG has been suggested in an attempt to inhibit Fcγ receptor-mediated platelet activation, since in the treatment of severe autoimmune HIT this treatment resulted in rapid increase in platelet count18,20. Unfortunately, platelet response of patient 1 to high-dose IVIG was striking but transient. Poor platelet increase was observed after three sessions of PEx. Early non-heparin anticoagulation, in addition to high-dose IVIG and steroids, is warranted in VITT to prevent thrombosis21,22 and, in view of the patient 1 clinical course, it should have been considered soon after thrombectomy in order to prevent reocclusion of the damaged vessel. However, early anticoagulation of patients affected by large brain infarct increases the risk of hemorrhagic transformation23. Moreover, the patient underwent decompressive craniectomy on day 3, which is an additional contraindication to early full-dose anticoagulation. Hence, we started with Fondaparinux 2.5 mg per day and we increased the dosage to 7.5 mg per day after 2 weeks from stroke onset.

Many scientific questions regarding VITT are still unanswered as highlighted by a recently published editorial24. Although limited to two patients, data collected in this report strengthen the hypothesis that platelets could be initially activated by unknown factors related to the ChAdOx1 nCov-19 vaccine/host interaction. An atypical autoimmune HIT may be the consequent event that could amplify platelet activation and coagulation cascade. The rare occurrence of VITT also in patients who received the Ad26.COV2.S vaccine (Johnson & Johnson/Janssen)7,8,9 suggests a possible pathogenic role of the adenoviral vector. In addition, a potential role for the ChAdOx1 nCov-19 vaccine cell-culture-derived proteins and components (i.e. EDTA) has been recently postulated25. More investigations are warranted to elucidate this and other hypotheses26 and clarify the pathogenesis of this catastrophic syndrome.

One limitation of the present paper is that we have not tested the PF4 antibody titer immediately after plasma exchange, and hence we do not know whether the recurrent thrombocytopenia was due to a persistent high level of PF4 antibody titer. Another limitation is that we have not dosed soluble DNA or citrullinated histones, markers of NETs, in the patients’ sera. NETs are released from the neutrophils in response to extracellular stimuli to contain infection, but their excessive formation can be detrimental by inducing microvascular thrombosis and propagating inflammation27. Greinacher et al.25 observed that VITT patient serum incubated with PF4 and purified neutrophils induces NETs formation (NETosis) with strongly enhanced response in the presence of platelets. NETosis could play a role in amplifying thrombi formation in VITT, but further studies are needed to confirm this issue.

In conclusion, our two cases of young adult women with massive brain artery thrombosis in addition to extensive systemic venous thrombosis, thrombocytopenia, and PF4–polyanion antibodies, developed within 10 days from ChAdOx1 nCov-19 vaccination, might represent a stroke variant of the recently named VITT syndrome. Ischemic stroke may be the first serious symptom of VITT. Physicians should be aware of this possibility and should carefully investigate patient medical history asking for any previous vaccination (within 4–28 days) especially in young female stroke patients. If low platelet count would be present (it could also be mild initially), a chest and abdominal CT scan should be performed to rule out venous thrombosis, together with an anti-PF4/heparin IgG enzyme immunoassay and functional platelet assay. If diagnosis of VITT is confirmed (but also if the suspect of VITT is high, without waiting for specific tests), non-heparin anticoagulation should be promptly started, if platelet count is up to 50 × 109/L, as well as therapy with high dose of steroids and IVIG. Platelet transfusion should be avoided.