Anti-PF4 mediated thrombocytopenia and thrombosis associated with acute cytomegalovirus infection displays both HIT-like and VITT-like characteristics

Abstract

Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is one of several anti-platelet factor 4 (anti-PF4)-associated immune thrombocytopenia and thrombosis (PITT) syndromes. As well as following adenoviral vector vaccines, VITT has recently been described following acute adenovirus infection. We describe a patient with PITT following acute cytomegalovirus infection. The antibody clonotype and PF4 epitopes were distinct from those identified in VITT, and they were detectable as a paraprotein. PITT should be considered in all patients with thrombocytopenia and thrombosis, even without preceding vaccination or heparin, but who otherwise meet the VITT criteria defined by the British Society of Haematology Expert Panel.

Keywords: FCY receptor; cytomegalovirus; heparin‐induced TP; monoclonal antibodies; platelet activation; platelet factor 4; thrombocytopenia; thrombosis.

FIGURE 1

Platelet function testing shows heparin‐induced thrombocytopenia (HIT)‐like, vaccine‐induced immune thrombocytopenia and thrombosis (VITT)‐like or a mixed picture depending on testing modality. Healthy donor platelet‐rich plasma (PRP) was stimulated with patient serum with/without heparin and/or platelet factor 4 (PF4) (A) in the presence of a fluorescein isothiocyanate (FITC)‐conjugated activation marker antibody (as denoted by the HITAlert™ Kit package insert). Percentage of platelets positive for P‐selectin was measured by flow cytometry (Ai). Chemiluminescence AcuStar™ HIT‐IgG and LIFECODES™ Anti‐PF4/Heparin HIT IgG enzymatic immunoassay (EIA) (Aii). C‐Serotonin loaded healthy donor washed platelets (3 ×10⁸/mL) were stimulated with patient serum with/without heparin and/or PF4 and/or IV.3. The supernatant was then analysed for radioactivity using a β‐counter (B). Healthy donor washed platelets (2 × 10⁸/mL) were stimulated with serum ± PF4 (10 μg/mL) ± Heparin (0.5 U/mL), IV.3 (10 μg/mL) or vehicle (PBS). Representative aggregation trace (C) and mean data (D) shown. Data presented as maximum aggregation (area under the curve; AUC/min). Mean + SD, n = 3–5, statistical analysis by one‐way analysis of variance (ANOVA). ****p < 0.0001.

FIGURE 2

Anti‐platelet factor 4 (anti‐PF4) epitope mapping and proteomic analysis show distinct PF4 binding sites and anti‐PF4 clonotypes between patients with vaccine‐induced immune thrombocytopenia and thrombosis (VITT) and this patient following cytomegalovirus infection. Antibodies show similar negatively charged antigen‐binding regions to those with VITT. Serum was added to ELISA plates coated with wild‐type or mutant PF4, and the degree of anti‐PF4 binding was assessed using an alkaline phosphatase‐conjugated secondary antibody as previously described (A). Residues previously identified as binding sites for PF4‐dependent VITT antibodies are shown in red, and those identified as binding sites for PF4‐independent VITT antibodies are shown in blue. Residues distinct from previously recognized sites are shown in yellow. Patients with VITT show a stereotypic clonotype featured by an identical immunoglobulin lambda variable 3‐21*02 (IGLV3‐21*02) light chain paired with a single heavy chain expressing a shared clonotypic ‘GLED’ amino acid motif. In comparison, this patient shows different immunoglobulin light and heavy chain molecular signatures. (IGLV, immunoglobulin lambda variable; LCDR3, light‐chain complementarity‐determining region 3; IGHV, immunoglobulin heavy variable; HCDR3, heavy‐chain complementarity‐determining region) (B). Paratope modelling for VITT and this patient's anti‐PF4 antibodies show a similar negatively charged antigen‐binding regions that electrostatically match the positively charged PF4 tetramer (C). The scale bar indicates surface electrostatic potential and corresponds to acidic (red) and basic (blue) amino acid residues. *indicates data previously shown in Wang et al. Blood 2022 and Wang et al. New Eng J Med 2024.,