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- vaccine-induced immune thrombotic thrombocytopenia (1)
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Background
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a prothrombotic, heparin-induced thrombocytopenia (HIT)-mimicking, adverse reaction caused by platelet-activating anti-platelet factor 4 (PF4) antibodies that occurs rarely after adenovirus vector-based COVID-19 vaccination. Strength of PF4-dependent enzyme immunoassay (EIA) reactivity—judged by optical density (OD) measurements—strongly predicts platelet-activating properties of HIT antibodies in a functional test. Whether a similar relationship holds for VITT antibodies is unknown.
Objectives
To evaluate probability for positive platelet activation testing for VITT antibodies based upon EIA OD reactivity; and to investigate simple approaches to minimize false-negative platelet activation testing for VITT.
Methods
All samples referred for VITT testing were systematically evaluated by semiquantitative in-house PF4/heparin-EIA (OD readings) and PF4-induced platelet activation (PIPA) testing within a cohort study. EIA-positive sera testing PIPA-negative were retested following 1/4 to 1/10 dilution. Logistic regression was performed to predict the probability of a positive PIPA per magnitude of EIA reactivity.
Results
Greater EIA ODs in sera from patients with suspected VITT correlated strongly with greater likelihood of PIPA reactivity. Of 61 sera (with OD values >1.0) testing negative in the PIPA, a high proportion (27/61, 44.3%) became PIPA positive when tested at 1/4 to 1/10 dilution.
Conclusions
VITT serology resembles HIT in that greater EIA OD reactivity predicts higher probability of positive testing for platelet-activating antibodies. Unlike the situation with HIT antibodies, however, diluting putative VITT serum increases probability of a positive platelet activation assay, suggesting that optimal complex formation depends on the stoichiometric ratio of PF4 and anti-PF4 VITT antibodies.
Platelet factor 4 (PF4, synonym: CXCL4) is an evolutionary old chemokine with proposed roles in hemostasis and antimicrobial defense. In addition, PF4 has attracted considerable attention as a crucial mediator of one of the most prothrombotic adverse drug effects affecting blood cells, heparin-induced thrombocytopenia (HIT). Interest in PF4 substantially increased in 2021 when it was identified as the target antigen in the life-threatening adverse effect, vaccine-induced immune thrombotic thrombocytopenia (VITT). We address the concept that a major biological function of PF4—a strongly cationic chemokine—is to bind to negatively-charged prokaryotic microorganisms, resulting in structural changes in PF4 that trigger a danger signal recognized by the adaptive immune system. Application of biophysical tools has provided substantial insights into the molecular mechanisms by which PF4 becomes immunogenic, providing insights into a new mechanism of autoimmunity. Binding of autoantibodies with high affinity induces conformational change(s) in the endogenous protein, which are then recognized as foreign antigen, as exemplified by the prothrombotic disorders, autoimmune HIT and VITT. The final part of our review summarizes current assays for HIT and VITT, explaining how structural aspects of anti-PF4 pathobiology relate to assay design and performance characteristics. Currently, functional (platelet activation) assays using washed platelets detect HIT antibodies when heparin is added, and VITT antibodies when PF4 is added. Solid-phase PF4-dependent immunoassays using microtiter plates are sensitive for both HIT and VITT antibodies, while rapid immunoassays, in which the PF4/heparin antigen is coated on beads, are sensitive and specific for HIT, but not for VITT antibodies.