Nucleic Acids Interfere with PF4 Tetramer Formation and Stabilise PF4 Monomers to Bind Non-Pathogenic HIT Antibodies: Subtypes of Non-Pathogenic Antibodies Emerge

Introduction

The pathogenesis of heparin-induced thrombocytopenia (HIT) primarily involves anti-platelet factor(PF)4/heparin antibodies engaging FcγRIIa receptors on platelets to trigger a deleterious cascade of activation, thrombosis, and thrombocytopenia. Recent work with murine monoclonal antibodies has shed light on epitopic determinants that potentiate platelet activation (pathogenic KKO vs non-pathogenic RTO antibodies), but these mechanisms may not fully reflect the gamut of polyspecific antibodies seen in human clinical practice. We examined the development and characteristics of anti-PF4/heparin antibodies in patients undergoing cardiac surgery.

Methods

After obtaining informed consent from patients undergoing cardiac bypass surgery, we performed ELISA using: (1) commercial Diagnostica Stago Asserachrom IgG kit (Cat.Nr 00624); or (2) in-house ELISA with platelet derived native(n)PF4. ELISA performed as per manufacturer instructions and published methods. Bacterial genomic nucleic acids (NA) were extracted from overnight culture of BL21(DE3)pLysS-wtPF4 using Promega kit Wizard® SV Genomic DNA Purification System (#A2361, Promega). Cross linking assays were performed as previously described using Bis(sulfosuccinimidyl)suberate (BS3) obtained commercially (#21585, Thermo Scientific Pierce). Optical density was normalised with controls to assist interassay comparability.

All samples were screened with the commercial kit day 7 post-operatively, or the nearest available serum between day 4-10. Positive samples were then examined longitudinally to chart the onset of antibody formation and their duration.

Results

1. Commercial ELISA identified 30/123 (24%) patients positive for anti-PF4/heparin antibodies following cardiac surgery, but there was no HIT observed in the complete cohort of 123 patients and therefore all 30 were false positive.

2. Surprisingly, in-house ELISA using nPF4/heparin was negative for 22/30, but the addition of NA to PF4 rescued the OD of these to become positive again.

3. In contrast, OD fell for all true HIT controls with the addition of NA, as they did for a subset of the false-positives 7/30 [Figure A].

4. Five patients had persistently raised OD despite the addition of NA, suggesting the presence of both NA/nPF4/heparin antibodies and nPF4/heparin antibodies.

5. Overall, the incidence of false-positive anti-PF4/heparin antibodies was only 12/123 (10%) using the in-house ELISA method with nPF4. SRA was performed on all patients with nPF4/heparin antibodies, but they were all negative.

6. Spiking nPF4 with increasing concentrations of NA caused reduction of tetramer formation and an increasing proportion of dimeric and monomeric forms seen on cross-linkage assays [Figures B and C].

7. Longitudinal studies demonstrated that all 13 patients positive by commercial ELISA with only low range absolute results (OD <0.4), and without increase at later timepoints, possessed only NA/nPF4/heparin antibodies.

8. By contrast, of the 14 patients whose ELISA OD increased over time, seven had evidence of nPF4/heparin antibodies. Three patients were ELISA positive pre-operatively, and only one of these had evidence of nPF4/heparin antibodies.

Conclusions

Increasing amounts of NA inhibit PF4 tetramer formation, and reduce the ability for HIT antibodies to bind. Thus, NA may participate in the preferential presentation of PF4 monomers that are recognised by non-pathogenic NA/PF4 monomer/heparin antibodies, similar to RTO. There is a smaller subset of "true" non-pathogenic antibodies that bind to PF4 tetramers, and an even smaller number of patients who may possess both classes of non-pathogenic antibodies. We speculate that pre-formed antibodies to NA/PF4 monomers are responsible for the majority of false positive results from commercial ELISA kits, and these are unrelated to the pathogenic anti-PF4/heparin antibodies of HIT.

Figure Caption

A: Representative example of five patients with increasing ELISA OD in the presence of NA (NA1-5), two patients with OD that fell in the presence of NA (TFP1-2), and known HIT serum with reduced OD in the presence of NA. B: Reduction in PF4 tetramer formation and increasing PF4 monomers with increasing NA concentration. Lanes from left-right: control PF4 without BS3, and then increasing concentration of NA from 0, 5, 30 and 45ng/μL with BS3. C: Percentage of PF4 as tetramers or monomers with increasing concentration of NA.

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