Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Study participants

Participants included patients diagnosed with VITT (n = 5), patients diagnosed with HIT (n = 10) and healthy volunteers (n = 10). VITT diagnosis was based on four criteria: recent AstraZeneca vaccination; positive for anti-PF4 IgG antibodies; positive in the PF4-enhanced SRA; and no previous exposure to heparin. HIT diagnosis was confirmed based on three criteria: the 4Ts score, in which all patients with HIT had a clinical score of at least 4; a positive commercially available PF4-enhanced heparin-dependent IgG/A/M-specific EIA (Immucor, OD ≥ 0.45) and a positive SRA (SRA ≥ 20% 14C-serotonin release)27. This study was approved by the Hamilton Integrated Research Ethics Board (HiREB) and informed written consent was obtained from all participants.

Platelet activation assays

Platelet activation assays were performed in the presence of heparin using the SRA, and including a modification in which increasing doses of exogenous PF4 were added, rather than heparin (PF4-SRA)27,28. Some assays were performed with high concentrations of unfractionated heparin (100 IU ml−1), or with an Fc-receptor-blocking monoclonal antibody (IV.3).

Epitope mapping of antibody binding to PF4 from VITT and HIT samples using alanine-scanning mutagenesis

The full-length DNA coding sequence of human PF4 was cloned into the pET22b expression vector using restriction sites NdeI and HindIII (GenScript). The PF4 mutants were expressed and purified as previously described5,29. In brief, PF4 mutants were designed whereby non-alanine amino acids in wild-type PF4 were mutated to alanine and the alanine amino acids in wild-type PF4 were mutated to valine. PF4 mutants were introduced into Escherichia coli ArcticExpress (DE3) cells (Agilent Technologies). For the overexpression of PF4 mutants, cultures were grown at 37 °C to mid-exponential phase before induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and grown at 37 °C for 3 h. E. coli cells for each wild-type PF4 or PF4 mutant were lysed by sonication in 20 mM sodium phosphate, pH 7.2, 400 mM sodium chloride, 1.4 mM β-mercaptoethanol, 5% (v/v) glycerol, 1% (v/v) Triton X-100 (Thermo Fisher Scientific), and 0.5% (w/v) sodium deoxycholate (MilliporeSigma) with 2 mM MgCl 2 , 10 μg ml−1 DNaseI (MilliporeSigma) and EDTA-free protease inhibitor cocktail (Roche). The supernatant was then cleared by centrifugation at 40,000g for 40 min at 4 °C and applied onto a HiTrap Q HP column (Cytiva Life Sciences) equilibrated with 20 mM sodium phosphate, pH 7.2, 400 mM sodium chloride, 1.4 mM β-mercaptoethanol and 5% (v/v) glycerol. The flow-through of the Q HP column was then stored at 4 °C, overnight. The following day, the serum was diluted twofold to yield a sodium chloride concentration of 200 mM with 20 mM sodium phosphate, pH 7.2, 1.4 mM β-mercaptoethanol and 5% (v/v) glycerol, syringe-filtered with a 0.2-μm filter (Acrodisc) and loaded onto a HiTrap Heparin HP column (Cytiva Life Sciences). Contaminants were eluted with 0.5 M sodium chloride and PF4 was eluted with a linear gradient from 0.5 to 2 M sodium chloride. Fractions containing pure wild-type or PF4 mutants were pooled, concentrated and buffer-exchanged to phosphate-buffered saline (PBS) and 1.5 M sodium chloride. The concentration of PF4 was determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Protein expression and purity was assessed for each PF4 mutant using 4–18% denaturing SDS–PAGE.

The effect of the 70 amino acids on the binding of anti-PF4–heparin antibodies in patient samples was measured and analysed in a similar manner to that described previously5. The binding of anti-PF4–heparin antibodies to wild-type PF4 and PF4 mutants was measured using a modified PF4–heparin IgG-specific EIA5,13. Microtitre well plates (384 wells; Thermo Scientific Nunc) were coated with 10 μg ml−1 streptavidin and 1 IU ml−1 biotinylated-heparin and blocked with PBS supplemented with 3% (v/v) bovine serum albumin (BSA) for 2 h at ambient temperature. Wild-type PF4 or PF4 mutants at 5 μg ml−1 were then added and incubated for 1 h at ambient temperature. Diluted patient samples (1:50 prepared in 1% BSA in PBS) in technical duplicates were added to the plates and incubated for 1 h at room temperature. After washing, alkaline-phosphatase-conjugated goat anti-human IgG (γ-chain-specific, Jackson ImmunoResearch Laboratories) was added at a 1:3,000 dilution and incubated for 1 h at ambient temperature. The addition of 1 mg ml−1 para-nitrophenylphosphate (MilliporeSigma) substrate dissolved in 1 M diethanolamine buffer (pH 9.6) was used for detection. The OD was measured at 405 nm and 490 nm (as a reference) using a BioTek 800TS microplate reader (BioTek) to assess the binding of antibodies to wild-type PF4 and PF4 mutants. Results were reported as a percentage of the loss of binding relative to wild-type PF4 binding.

Heparin inhibition of VITT anti-PF4 antibodies

Microtitre well plates (96 wells, Nunc Maxisorp) were coated overnight at 4 °C with 100 μl per well of PF4 (60 μg ml−1) diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6). The plates were then blocked with 200 μl per well of 3% (v/v) BSA prepared in PBS at room temperature for 2 h. VITT samples (n = 4) that were available were diluted 1:50 with 1% BSA in PBS and pre-incubated with increasing concentrations of unfractionated heparin (final concentrations of 0.1, 0.5, 1, 5 and 100 IU ml−1; Pfizer) for 1 h at room temperature. The blocking solution was removed from the microtitre well plates and the VITT samples and heparin mixtures (100 μl per well) in technical duplicates were added to the plates, which were then incubated for 1 h at room temperature. The plates were washed twice with PBS–0.05% Tween 20 and three times with PBS. Bound human IgG antibodies were detected with 100 μl per well of alkaline-phosphatase-conjugated goat anti-human IgG (γ-chain-specific, 1:3,000, Jackson Immuno-Research Laboratories) antibody prepared in 1% (v/v) BSA in PBS. Plates were washed as before and followed with the addition of 100 μl substrate (para-nitrophenylphosphate dissolved in diethanolamine buffer (MilliporeSigma)). The OD was measured at 405 nm and 490 nm (as a reference) using a BioTek 800TS microplate reader (BioTek).

Purification of total IgG antibodies

Total IgG antibodies from two VITT samples were purified for further analysis of binding kinetics. A volume of 2 ml protein G-coated sepharose beads (Thermo Fisher Scientific) was washed three times with PBS at room temperature, 300g for 5 min. Patient samples were heat-inactivated at 56 °C for 30 min and diluted three times in PBS. The samples were then transferred to protein G sepharose beads and incubated at room temperature for 1 h before extensive rinsing with 30 ml PBS. Total IgG was eluted from the protein G sepharose beads with 0.1 M glycine, pH 2.7 and neutralized by Tris buffer, pH 8.0.

Binding kinetics of VITT and HIT antibodies using BLI

Wild-type PF4 was labelled with biotin as previously described30. In brief, wild-type PF4 and PF4 mutants were incubated with 5× the volume of heparin sepharose 6 fast flow affinity chromatography medium (Cytiva Life Sciences) for 1 h with shaking at ambient temperature. EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) was added to the PF4 and heparin sepharose mixture in 20 molar excess and allowed to react for 1 h with shaking at ambient temperature. The biotinylated wild-type PF4 or PF4 mutants were eluted from the heparin sepharose using PBS and 2 M sodium chloride. Absorbance at 280 nm was measured using a spectrophotometer (Eppendorf AG) and used to calculate the concentration. Biotinylation of PF4 was checked using a streptavidin-coated anti-PF4–heparin EIA.

BLI experiments were performed using the Octet Red 96 instrument (FortéBio). Samples or buffer were dispensed into 96-well black flat-bottom microtitre plates (Greiner Bio-One) diluted in PBS supplemented with 1% (v/v) BSA at a volume of 200 μl per well with an operating temperature maintained at 23 °C. Streptavidin-coated biosensor tips (FortéBio) were hydrated with 1% BSA in PBS (MilliporeSigma) to establish a baseline before antigen immobilization for 60 s. Biotinylated recombinant PF4 (final concentration 7.5 μg ml−1), alone or complexed with 0.125 IU ml−1 unfractionated heparin (LEO Pharma), was then immobilized on the biosensor tips for 1,200 s at 1,000 rpm followed by a baseline re-establishment for 1,800 s at 1,000 rpm. Antigen-coated sensors were then reacted with heat-inactivated patient samples or purified total IgG at a 1:32 dilution in duplicate for 780 s at 1,000 rpm followed by a dissociation step for 3,000 s at 1,000 rpm. Data were analysed using Octet User Software v.3.1 using the 2:1 heterogenous ligand-binding model. Reference values from control wells with buffer alone were subtracted and all results were aligned to the measured baseline. The binding profile response of each sample was expressed as the average wavelength shift in nm.

Data acquisition, statistical analysis and reproducibility

Differences between data were tested for statistical significance using the paired or unpaired t-test and the Mann–Whitney test. P-values are reported as two-tailed and P < 0.05 was considered to be statistically significant. All statistical analyses were conducted using GraphPad Prism v.9.1.0 (GraphPad Software). Experiments were repeated with technical duplicates independently twice with similar results unless otherwise stated.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.