ABSTRACT
Introduction Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare, but severe side effect after Covid-19 and other vaccinations. First cases of VITT-mimicking antibodies in unvaccinated patients with recurrent thrombosis have been described. Differentiation between heparin-induced thrombocytopenia (HIT) and VITT is difficult in some patients. Widely used enzyme-linked immunoassays (EIA) cannot differentiate between the two, some of them even fail to detect VITT antibodies. So far, differentiation between HIT-like and VITT-like anti-PF4 antibodies can only be performed in specialized laboratories by functional tests using the heparin-induced platelet activation (HIPA) or PF4-induced platelet activation (PIPA) test. We have developed an assay, which can distinguish between HIT and VITT antibodies and can be used in any hospital laboratory. Method Confirming platelet-activation assays (HIPA and PIPA) were performed as described.[1] We defined 3 cohorts: 1) Negative controls (n = 112, including 35 healthy donors from before 2020, 46 clinical patients suspected for HIT but with negative EIA and HIPA and 31 non-thrombotic patients);2) classical HIT-patients with positive EIA and HIPA (n = 121);3) typical VITT patients (n = 63;presenting after vaccination with adenoviral vector-based Covid-19 vaccine and positive EIA and PIPA). Samples were analyzed by an automated coagulation analyzer ACL AcuStar (Werfen / IL Inc., Bedford, MA, USA) using HemosIL AcuStar HIT-IgG(PF4-H) and a prototype of VITT-IgG(PF4) assay according to the manufacturer's protocol. For both assays, raw data was analyzed as relative light units (RLU). Results All VITT samples were positive in the prototype VITT-assay (Fig. 1);only a few (n = 9;14.3 %) also showed weakly positive results in the HIT-assay. On the other hand, most of the HIT samples showed positive results in the HIT-assay (113;93.4 %), 34 of them (30.1 %) also reacted positive in the prototype VITT-assay (12 of them strongly;10.6 %), and three demonstrated an antibody pattern like autoimmune VITT. Negative control samples where all non-reactive in the HITassay and served to adjust the cutoff for the prototype VITT-assay. Conclusion The different reaction pattern of samples of HIT and VITT patients using HemosIL AcuStar HIT-IgG(PF4-H) and a VITT prototype assay was able to distinguish between the two antibody entities for the first time. The combination of assays can facilitate a rapid decision whether heparin may be used for treatment and also identify patients with autoimmune-VITT as a cause of recurrent thrombosis. (Table Presented).
ABSTRACT
Background: SARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that-like autoimmune heparin-induced thrombocytopenia-is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies. Aims: To understand how the SARS-CoV-2 vaccine ChAdOx1 nCov-19 can induce anti-PF4 antibodies and how these antibodies induce thrombosis Methods: We investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using 3D-super-resolution microscopy, dynamic light scattering, and transmission electron microscopy. Vaccine composition was analyzed by mass spectrometry. Experimental vascular leakage models assessed early post-vaccine reactions. We evaluated VITT antibody-mediated platelet activation and formation of procoagulant DNA-containing neutrophil extracellular traps (NETs), including within VITT patient cerebral venous thrombi. Results: Biophysical analyses showed HEK cell line proteins and free virus proteins in the vaccine, formation of complexes between PF4 and vaccine constituents (including viral proteins) that were recognized by VITT antibodies. In a murine model, EDTA (vaccine constituent) increased microvascular leakage with dissemination of virus-and cell culture-derived human proteins. Free viral proteins and preexisting antibodies in normal human sera reacting with vaccine-containing constituents, likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. PF4-binding polyanions (polyphosphates, DNA) enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven NETs formation;further evidence for NETosis included elevated NETs biomarkers and low DNase activity in VITT sera, and NETs/ neutrophil-rich cerebral vein thrombi extracted from VITT patients. Conclusions: ChAdOx1 nCoV-19 vaccine constituents form antigenic complexes with PF4, and EDTA increases microvascular permeability, enhancing risk of early post-vaccination acute inflammatory reactions;PF4/polyanion antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. Resulting high-titer anti-PF4 antibodies activate platelets and induce neutrophil activation with NETosis, further fueling the VITT prothrombotic response.