Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin‐induced thrombocytopenia

Abstract

Many patients produce antibodies but few lead to heparin‐induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non‐pathogenic antibodies. Summary BackgroundHeparin‐induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti‐PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. ObjectivesTo identify PF4 amino acids essential for binding pathogenic HIT antibodies. MethodsAlanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet‐activating murine monoclonal anti‐PF4/heparin antibody (KKO) and HIT patient sera (n = 9). Results and ConclusionsWe identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n = 45) and non‐pathogenic antibodies (EIA‐positive but not platelet activating, n = 28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4.

Reactivity of platelet‐activating and nonplatelet‐activating anti‐PF4/heparin antibodies in enzyme immunosorbent assays under different conditions

“…Although the conditions shown here are obviously not suitable for clinical application, others may take advantage of these basic data to improve antigen‐based assays for aPF4/H Abs to better discriminate between platelet‐activating and nonplatelet‐activating aPF4/H Abs. Potential approaches beyond the experiments described here could be to combine changes in pH and salt with recombinant PF4 mutants and/or the use of different polyanions to generate complexes with PF4 . CD spectroscopy can be used as a tool to identify the magnitude of conformational changes of PF4 in such designed PF4/polyanion complexes as an approximation to identify test conditions for further assessment with clinical antibody samples.…”

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia

“…Previous studies have shown that the binding epitopes of HIT antibodies are non-contiguous and conformation-dependent 13 . Therefore, to identify the specific amino acid targets of VITT antibodies on PF4, we used alanine scanning mutagenesis and produced 70 unique recombinant PF4 mutants, each differing by a single amino acid 5 . We defined a critical binding amino acid as a corresponding PF4 mutant that caused a greater than 50% reduction in binding compared to wild-type PF4.…”

Hypotheses behind the very rare cases of thrombosis with thrombocytopenia syndrome after SARS-CoV-2 vaccination

“…While the epitopes involved in the binding of classical HIT antibodies to PF4 are partially known,[ 47 , 48 ] those involved in autoimmune HIT syndromes have not yet been documented, and might be very different. [ 49 ] However, it is not excluded that these IgG antibodies directed against other epitopes are also able to activate platelets. In addition, the fact that these cases were mainly reported in young women should question on the possibility that we are facing previously immunized women.…”