Monoclonal Antibodies in the Pathogenesis of Heparin-Induced Thrombocytopenia

Abstract

Background: Heparin-induced thrombocytopenia (HIT) is an immune-mediated platelet disorder caused by antibodies that target complexes of platelet factor 4 (PF4) and heparin. HIT has been characterized as a polyclonal immune response; however, studies of other rare anti-PF4 disorders have identified clonally restricted antibodies.

Methods: In this study, we investigated the clonality of pathogenic HIT antibodies. Antibodies against PF4-heparin were affinity-purified with the use of PF4-heparin beads from serum samples obtained from nine patients with clinically and serologically confirmed HIT. Antibody clonality was assessed by means of immunofixation electrophoresis and mass spectrometry. Antibody binding to PF4 was evaluated by an enzyme immunoassay, and functional platelet activation was evaluated with the use of a P-selectin expression assay. HIT antibody epitopes were mapped in two patients with the use of a PF4 mutant library.

Results: Serum samples from all nine patients with HIT were positive for platelet-activating antibodies against PF4-heparin by enzyme immunoassay, as well as the P-selectin expression assay, and six samples (67%) had a monoclonal antibody detectable by immunofixation electrophoresis. The affinity-purified antibodies against PF4-heparin from all nine samples activated platelets in the P-selectin expression assay, and mass spectrometry showed monoclonality. After affinity purification, antibody-depleted serum samples lost binding activity in the enzyme immunoassay and functional activity in the P-selectin expression assay, which confirmed the removal of the pathogenic antibodies. The epitopes on PF4 targeted by anti-PF4-heparin antibodies from serum samples were the same as those targeted by the affinity-purified monoclonal antibodies.

Conclusions: The pathogenic antibodies in all nine patients with HIT were found to be monoclonal. This finding provides insight into the pathogenesis of HIT and has implications for improved diagnostics and targeted therapeutics. (Funded by the Canadian Institutes of Health Research and the National Institutes of Health.).

Figure 1.

Detection and platelet-activating function of anti–PF4/heparin antibodies in purified HIT serum fractions. (A) An anti-PF4/heparin IgG/A/M EIA was used to determine the level of anti-PF4/heparin antibodies in HIT patients (n=9). EIA results for affinity-purified fractions were compared with serum and antibody-depleted serum. (B) Functional activity of anti–PF4/heparin antibodies were assessed using the PEA. Platelet activation under various conditions (buffer alone, PF4, PF4 + 125 U/mL heparin, and PF4 + IV.3) was normalized to TRAP-6. Error bars represent ± standard deviation. (C-E) Determining clonality of pathogenic anti-PF4/heparin antibodies in HIT patients. MS analysis of the affinity-purified fraction from a representative HIT sample that screened positive for an M-protein. (C) Mass distribution of the light chains showed a single sharp peak at 22.8 kDa, corresponding to a single polypeptide of the lambda isotype. (D) Mass distribution of the F(ab’)₂ fragment demonstrated a single peak, confirming antibody monoclonality. (E) Mass distribution of the heavy chains showed multiple peaks representing N-glycosylated polypeptides with varying glycan chain maturity, characteristic of IgG monoclonal antibodies.

Figure 2.

Purified monoclonal anti–PF4/heparin antibodies (green) from two M-protein–positive HIT patients were mapped on PF4 and compared with their original serum samples (green). Affinity-purified fractions showed overlap with serum epitope maps at 10 of 11 (90.9%) and 7 of 10 (70.0%) PF4-binding residues, respectively. VITT (red) and KKO monoclonal antibody (blue) binding sites are shown for comparison.