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

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.

Keywords

platelet factor 4 - monoclonal antibodies - heparin - thrombocytopenia

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.

Publication History

Received: 09 March 2020

Accepted: 13 August 2020

Article published online:
21 October 2020

• References

• 1 Greinacher A. Heparin-induced thrombocytopenia. N Engl J Med 2015; 373 (19) 1883-1884
• 2 Kreimann M, Brandt S, Krauel K. et al. Binding of anti-platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4. Blood 2014; 124 (15) 2442-2449
• 3 Arepally GM, Ortel TL. Heparin-induced thrombocytopenia. Annu Rev Med 2010; 61: 77-90
• 4 Greinacher A, Selleng K, Warkentin TE. Autoimmune heparin-induced thrombocytopenia. J Thromb Haemost 2017; 15 (11) 2099-2114
• 5 Padmanabhan A, Jones CG, Bougie DW. et al. Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis. Blood 2015; 125 (01) 155-161
• 6 Nguyen TH, Medvedev N, Delcea M, Greinacher A. Anti-platelet factor 4/polyanion antibodies mediate a new mechanism of autoimmunity. Nat Commun 2017; 8: 14945
• 7 Pouplard C, Amiral J, Borg JY, Vissac AM, Delahousse B, Gruel Y. Differences in specificity of heparin-dependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium. Br J Haematol 1997; 99 (02) 273-280
• 8 Arepally GM, Kamei S, Park KS. et al. Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies. Blood 2000; 95 (05) 1533-1540
• 9 Nguyen TH, Greinacher A. Platelet factor 4/heparin complexes present epitopes differently on solid-phase vs platelet surfaces. Blood 2017; 129 (26) 3498-3501
• 10 Kizlik-Masson C, Vayne C, McKenzie SE. et al. 5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. J Thromb Haemost 2017; 15 (10) 2065-2075
• 11 Pouplard C, Leroux D, Rollin J, Amiral J, May MA, Gruel Y. Incidence of antibodies to protamine sulfate/heparin complexes incardiac surgery patients and impact on platelet activation and clinical outcome. Thromb Haemost 2013; 109 (06) 1141-1147
• 12 Juhl D, Eichler P, Lubenow N, Strobel U, Wessel A, Greinacher A. Incidence and clinical significance of anti-PF4/heparin antibodies of the IgG, IgM, and IgA class in 755 consecutive patient samples referred for diagnostic testing for heparin-induced thrombocytopenia. Eur J Haematol 2006; 76 (05) 420-426
• 13 Cattaneo M, Cerletti C, Harrison P. et al. Recommendations for the standardization of light transmission aggregometry: a consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost 2013; DOI: 10.1111/jth.12231.
• 14 Greinacher A, Amiral J, Dummel V, Vissac A, Kiefel V, Mueller-Eckhardt C. Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay. Transfusion 1994; 34 (05) 381-385
• 15 Nguyen TH, Greinacher A, Delcea M. Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds. Nanoscale 2015; 7 (22) 10130-10139
• 16 Nguyen TH, Steinbock LJ, Butt HJ, Helm M, Berger R. Measuring single small molecule binding via rupture forces of a split aptamer. J Am Chem Soc 2011; 133 (07) 2025-2027
• 17 Krauel K, Weber C, Brandt S. et al. Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes. Blood 2012; 120 (16) 3345-3352
• 18 Merkel R, Nassoy P, Leung A, Ritchie K, Evans E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 1999; 397 (6714): 50-53
• 19 Evans E. Probing the relation between force--lifetime--and chemistry in single molecular bonds. Annu Rev Biophys Biomol Struct 2001; 30: 105-128
• 20 Evans E, Leung A, Hammer D, Simon S. Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force. Proc Natl Acad Sci U S A 2001; 98 (07) 3784-3789
• 21 Webb B, Sali A. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics 2016; 54: 1
• 22 Cai Z, Yarovoi SV, Zhu Z. et al. Atomic description of the immune complex involved in heparin-induced thrombocytopenia. Nat Commun 2015; 6: 8277
• 23 Bourquard T, Musnier A, Puard V. et al. MAbTope: a method for improved epitope mapping. J Immunol 2018; 201 (10) 3096-3105
• 24 Sachais BS, Litvinov RI, Yarovoi SV. et al. Dynamic antibody-binding properties in the pathogenesis of HIT. Blood 2012; 120 (05) 1137-1142
• 25 Rauova L, Poncz M, McKenzie SE. et al. Ultralarge complexes of PF4 and heparin are central to the pathogenesis of heparin-induced thrombocytopenia. Blood 2005; 105 (01) 131-138
• 26 Bui VC, Nguyen TH. The role of single-molecule force spectroscopy in unraveling typical and autoimmune heparin-induced thrombocytopenia. Int J Mol Sci 2018; 19 (04) E1054
• 27 Cines DB, Yarovoi SV, Zaitsev SV. et al. Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia. Blood Adv 2016; 1 (01) 62-74
• 28 Amiral J, Pouplard C, Vissac AM, Walenga JM, Jeske W, Gruel Y. Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation. Br J Haematol 2000; 109 (02) 336-341