Thromb Haemost
DOI: 10.1055/s-0040-1717078
Cellular Haemostasis and Platelets

Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia

Caroline Vayne*
1  EA7501 GICC, University of Tours, Tours, France
2  Department of Haemostasis, Regional University Hospital Centre Tours, Tours, France
,
Thi-Huong Nguyen*
3  Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
4  Institute for Bioprocessing and Analytical Measurement Techniques, Heilbad Heiligenstadt, Germany
,
Jérôme Rollin
1  EA7501 GICC, University of Tours, Tours, France
2  Department of Haemostasis, Regional University Hospital Centre Tours, Tours, France
,
Noémie Charuel
1  EA7501 GICC, University of Tours, Tours, France
,
Anne Poupon
5  PRC, INRA, CNRS, University of Tours, Nouzilly, France
6  MAbSilico SAS, Nouzilly, France
,
Claire Pouplard
1  EA7501 GICC, University of Tours, Tours, France
2  Department of Haemostasis, Regional University Hospital Centre Tours, Tours, France
,
Nicole Normann
3  Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
,
Yves Gruel**
1  EA7501 GICC, University of Tours, Tours, France
2  Department of Haemostasis, Regional University Hospital Centre Tours, Tours, France
,
Andreas Greinacher**
3  Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
› Author Affiliations
Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation (NG 133/1–2) and the Institut pour la Recherche sur la Thrombose et l'Hémostase (IRTH).

Abstract

Background Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent “autoimmune” HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs.

Objectives To characterize 1E12, 1C12, and 2E1 in comparison to the heparin-dependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3).

Methods Interactions of Abs with PF4 and PF4/H were studied by enzyme-linked-immunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method).

Results 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin.

Conclusion 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs.

Authors' Contributions

C.V. and T.H.N. performed and designed the research, analyzed the data, and wrote the manuscript. J.R., A.G., and Y.G. designed the research, analyzed the data, and wrote the manuscript. C.P. analyzed the data and wrote the manuscript. N.C., A.P., and N.N. performed the research and analyzed the data. All authors reviewed and approved the final version of the manuscript.


* C.V and T.H.N. contributed equally to the study.


** Y.G and A.G. contributed equally to the design of this study.


Supplementary Material



Publication History

Received: 09 March 2020

Accepted: 13 August 2020

Publication Date:
21 October 2020 (online)

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