Emerging evidence suggests that platelet activation in VITT is mediated by the production of anti-PF4/polyanion antibodies followed by generation of IgG/PF4/polyanion immunocomplexes, massive neutrophil activation and release of NETs, endothelial activation and eventually diffuse venous and arterial thrombosis.15,24 Although the adenoviral vector has been proposed as the first trigger of platelet activation, this hypothesis is still unproven. Here, we provide evidence for an alternative mechanism that may generate a cascade of events culminating in full-blown VITT.
We have detected the Sars-cov-2 SP in serum of three VITT patients within 72 h from the index thrombotic event up to 35 days from vaccination, suggesting that these patients might be experiencing a prolonged/excessive production of the SP. We also observed that the SP co-localized with platelets in sections of the “white” arterial thrombus retrieved by Patient 1. As far as we know, this is the first evidence of SP inside a retrieved thrombus in VITT.
It has been demonstrated that Sars-cov-2 and the SP alone can directly activate platelets via ACE2/SP interaction and that platelet activation is suppressed by the recombinant human ACE2 protein and anti-SP monoclonal antibody.18 Accordingly, we have found a partial suppression of platelet integrin activation in all VITT patients by using an antibody directed against the Sars-cov-2 spike protein S1 subunit. Since stimulation of platelets by SP leads to PF4 release,18 it could then trigger platelet activation by forming IgG/PF4/polyanion immunocomplexes in individuals that express high levels of andi-PF4 antibodies.
A progressive reduction of ACE2 expression on platelets of critically ill COVID-19 patients and on platelets incubated with the Sars-cov-2,18 as well as on alveolar epithelial cells incubated with purified Sars-cov recombinant SP,25 has been observed. This evidence suggest a possible internalization and subsequent degradation18 or cleavage (or shedding)26,27 of the ACE2 receptor induced by the Sars-cov-2 and purified SP. We found ACE2 protein expressed on platelets of non-vaccinated HC, but not on those collected from a VITT patient, and ACE2 expression level was lower on platelets inside the VITT thrombus compared with a pre-pandemic control thrombus. Although limited to few subjects, these observations suggest a possible ACE2 receptor down-regulation on platelets in VITT. We detected sACE2 in serum from all three VITT patients with the highest levels in patient 3 who suffered from hypertension and used to take an ACE-inhibitor medication. Levels of ACE2, the main host cell receptor for the Sars-cov2, in human body is probably genetically determined.28 Besides its membrane form, which drives the virus into the host cell, a soluble ACE2 is well known, in part resulting from proteolytic cleavage and ectodomain shedding.26,27 Recently, although high levels of sACE2 have been observed in critically ill COVID-19 patients, a possible neutralizing and protective effect of sACE2 on virus spreading into the bloodstream has been hypothesized,29,30 and clinical trials in COVID-19 with human recombinant soluble ACE2 are ongoing (https://www.clinicaltrials.gov; NCT04287686). In this regard, it is interesting that only patient 3, whose sACE2 levels were quite high, recovered from VITT.
Based on our data we postulate a multiple-hit model for platelet activation in VITT aetiopathogenesis. The first hit being the direct interaction between the Sars-cov-2 SP and the ACE2 receptors on platelets and endothelial cells. Activated endothelial cells would induce platelet recruitment and adhesion by exposing adhesion receptors and releasing VWF. Activated platelets would release their granular contents which include large amounts of PF4 and enable the formation of immune complexes in individuals expressing high levels of anti-PF4 antibodies. The second hit being the stimulation of FcgRIIA by IgG/PF4/polyanion immune complexes resulting in the amplification of platelet activation; the third hit being the stimulation of neutrophils by IgG/PF4/polyanion immune-complexes and platelets, resulting in the release of NETs which in turn stimulates coagulation and platelets.15,24 Interestingly, patient 2 whose clinical course was rapidly fatal showed the highest level of NETs. A support to this 3-hit hypothesis is also the finding that despite the negativity of anti-PF4 antibodies at 35 days from admission, patient 3 serum continued to activate healthy donors’ platelets and still had detectable levels of soluble SP in the serum.
Some observations on vaccinated HCs need consideration. Unexpectedly, we found SP in serum of 2 out of 7 vaccinated HCs within 16 days from the first dose of ChAdOx1 nCoV-19. Both of them presented also detectable levels of sACE2. Neither of these two controls presented anti-PF4 antibodies, nor their sera could activate platelets from healthy donors. We can hypothesize that soluble SP variants capable of activating platelets are produced very rarely from alternative splicing events. A lower ACE2 receptor expression on platelets, possibly genetically-determined, could also justify the different chance of platelets to be activated by soluble SP. Other authors have found the full length SP and S1 subunit in plasma of respectively 3/13 and 11/13 healthy subjects who received the first dose of mRNA-1273 vaccine (Moderna),31 but clearance of both proteins correlated with IgG and IgA production. The clinical significance of this and our finding in HC is still unknown and more studies are warranted, but it seems to represent a harmless phenomenon.
One limitation of our study is the small number of participants. A second limitation is that we have not quantified the Sars-cov-2 nuclear antigen in serum to rule out a possible Sars-cov-2 infection, nor performed a Sars-cov-2 RNA in situ hybridization in addition to immunohistochemistry on thrombus of patient 1. However, all three patients were assessed to be negative for COVID-19 infection by rRT-PCR test on nasopharyngeal swab and patient 2 also on bronchoalveolar lavage. Serum antibodies to SP (IgG) were positive only in patient 1 suggesting an asymptomatic previous infection.
In conclusion, the present data provides first evidence that 1) DNA vaccine-encoded Sars-cov-2 soluble SP is detectable in VITT patients’ serum up to several weeks post-vaccination; 2) SP appears to co-localize with platelets in a thrombus retrieved from a VITT patients; 3) ACE2 expression on platelets seems to be reduced in VITT patients; 4) anti-SP antibody can partially inhibit platelets activated by VITT sera. All this data suggest that SP may be one of the platelet activation triggers in VITT via binding to ACE2 receptors expressed on platelets, and in part on endothelium. Anti-PF4/polyanion antibodies development could represent an epiphenomenon, which amplifies platelet aggregation, NETosis and coagulation cascade. This data needs to be confirmed by studies on larger samples of subjects.