Monoclonal Antibodies in the Pathogenesis of Heparin-Induced Thrombocytopenia

Author(s): Jared Treverton, B.Sc.^1,2; Donald M. Arnold, M.D.^2,3; Daniil G. Ivanov, Ph.D.⁴; Nikola Ivetic, Ph.D.^2,3; Yi Zhang, M.Sc.^2,3; Hossam A. Ali, B.Sc.⁴; Rumi Clare, B.Sc.^2,3; Igor A. Kaltashov, Ph.D.⁴; John G. Kelton, M.D.^2,3; Ishac Nazy, Ph.D.^1,2,3;

Source: N Engl J Med 2025;393:879-886

Dr. Anjan Patel's Thoughts

This NEJM study challenges the long-held view that heparin-induced thrombocytopenia (HIT) is a polyclonal antibody disorder. Using mass spectrometry and immunofixation, the investigators found that all 9 patients with HIT had monoclonal, platelet-activating anti–PF4–heparin antibodies, with 67% showing a detectable M-protein by immunofixation. Functional assays confirmed that only the monoclonal fraction was pathogenic. It looks like HIT is driven by a single rogue antibody clone, which could have big implications for diagnostics and targeted therapy—this would be a paradigm shift for how we think about this disease.

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.)

Author Affiliations

¹Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada; ²Michael G. DeGroote Centre for Transfusion Research, McMaster University, Hamilton, ON, Canada; ³Department of Medicine, McMaster University, Hamilton, ON, Canada; ⁴Department of Chemistry, University of Massachusetts, Amherst, Amherst

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