The association between thrombocytopenia and thrombosis, with a catastrophic clinical picture demonstrating both venous and arterial thromboses, raised rapidly the attention of clinicians. It was soon clear that we were facing a new disease, probably associated with a trigger linked to Adenovirus-Based SARS-CoV-2 Vaccines. The fact that these clinical pictures were not observed after mRNA vaccines put adenoviruses in the dock. The subsequent reports of similar cases associated with Janssen Vaccine, reinforced this hypothesis.

The binomial constituted by thrombocytopenia and thrombosis is very rare in medicine. We can recognize it in (a) Thrombotic thrombocytopenic purpura–together with anemia and hemolysis signs; (b) Heparin-induced thrombocytopenia and autoimmune HIT; (c) Catastrophic Antiphospholipid Syndrome [13]; (d) disseminated intravascular coagulation—together with signs of clotting factors consumption. The latter, however, represents a condition which can complicate several diseases associated with clotting activation, and does not represent a ‘per se’ disease.

Quickly it emerged the HIT hypothesis as the most plausible one. The time window for symptoms onset—between 4 and 16 days—comparable to that seen in HIT corroborated the hypothesis.

HIT is a rare immune-mediated adverse drug reaction occurring after exposure to heparin [14]. It is a serious and potentially fatal condition, which may be associated with the development of arterial or venous thrombotic events. From a pathophysiologic point of view, circulating molecules of heparin bind to platelet factor 4 (PF4), a plasmatic positively charged protein released upon platelet activation. PF4 normally binds to negatively charged glycosaminoglycans on the endothelium, displacing anti-thrombin and thus activating coagulation. However, PF4 binds with greater affinity to heparin/PF4 complexes, which become “neoantigens” and induce the formation of antibodies against them. Heparin-PF4-IgG immune complexes activate platelets through their FcγRIIA receptors, causing their activation, aggregation and the additional release of PF4, thus determining a positive feedback loop of further platelet activation. Pre-test clinical probability, screening for anti-PF4/heparin antibodies and documentation of their platelet activating capacity are the cornerstones of diagnosis. Both immunoassays—to detect the presence and quantify the titer of PF4-heparin antibodies—and functional assays—to assess the ability of anti-PF4-heparin antibodies to bind and activate platelets in the presence of heparin, are needed to confirm the diagnosis.

The time window for symptoms onset, the presence of both thrombocytopenia and thrombosis, with venous and arterial localizations, are the points which are similar between HIT and VITT. In the first cases described in Europe, the lethality was very high (about 20%) differently from that we usually observe in HIT. Furthermore, in a significant proportion of patients with VITT, the disease was complicated by DIC, with the presence of very low levels of platelet count (in the majority of cases < 20,000/mmc). These characteristics make this clinical entity more similar to autoimmune HIT with respect to classical HIT. Autoimmune HIT [15], in fact, is often complicated by DIC and it is characterized by a very low platelet count. Autoimmune HIT is the term adopted to define form of HIT in which the previous exposure to heparin is not always present: in these cases, we can demonstrate the presence of both heparin-dependent and heparin-independent antibodies activating platelets. In autoimmune HIT, some case reports [16] document the clinical efficacy of high-dose IgG administration which, conversely, is not routinely used in classical HIT. PF4 also binds to polyanions expressed on the cell wall of a wide variety of Gram-negative and Gram-positive bacteria and plays a role in bacterial host defense. Binding of PF4 to polyanions results in its conformational change. These antibodies are associated in the general population with the presence of chronic bacterial infections such as periodontal disease [17]. There is a continuum of clinical sequelae resulting from anti-PF4/polyanion antibodies (anti-PF4/P-ABS): the majority of individuals remain asymptomatic, while in HIT, anti-PF4/P-ABS activate platelets and the clotting system, resulting in life-threatening thrombotic complications when patients receive the polyanion heparin. Anti-PF4/P-ABS are also induced after major surgery without heparin treatment when mechanical compression devices are used for thrombosis prophylaxis [18]. In this case, continuous tissue compression likely acts as danger signal.

The extreme sequela is autoimmune HIT, in which individuals develop multiple vessel occlusions without any drug exposure. Typically, in these patients, PF4/P-ABS are found by enzyme-linked immunoassay (EIA) at very high titers and activate platelets in the absence of polyanions in functional assays.

On April 9, 2021, two papers were published reporting two EU case series of patients affected by this condition, named for the first-time VITT [4, 5]. In both papers, authors were able to document the presence of circulating antibodies able to activate platelets.

Two very recently published paper brought some more knowledge about the plausible sequence of events leading to VITT.

Mc Gonagle and Colleagues suggested that the local tissue micro-trauma following vaccine inoculation brings adenoviral DNA in contact with PF4, with substantially increased anti-PF4 autoantibody production in susceptible subjects [19]. The high-titer circulating autoantibodies against PF4 may lead to immuno-thrombosis in specific sites of susceptibility, such as the portal and the cerebral vein circulation, where an extensive platelet factor 4 (PF4)-viruses/microbiota interaction is already existing to ensure the normal immunity and antigen clearance.

Another postulated mechanism is based on the availability of soluble Spike protein variants in a systemic fashion as a result of alternative splice events [20]. These soluble Spike variants can bind to ACE2 expressing endothelial cells, which in turn may trigger an Antibody Dependent Cell-mediated Cytotoxicity (ADCC) or Complement-Dependent Cytotoxicity (CDC).

It can be assumed that this pathological phenomenon most probably will take place in those vessels where such soluble Spike protein variants can accumulate because of a non-unidirectional blood flow, i.e. the cerebral venous sinuses [20].

Whatever the pathogenic mechanism of VITT is, in order to document and diagnose this rare syndrome, besides clinical features (thrombocytopenia plus thrombosis with multiple localizations, mainly cerebral vein thrombosis and splanchnic thrombosis), laboratory should demonstrate the presence of antibodies anti-PF4 and, possibly, the ability of activating platelets. Unfortunately, not all immunological assays for detecting Ab anti-PF4/heparin have the same ability to detect also these VITT antibodies (6) and the administration of i.v. IgG might mask the functional ability of activating platelets.