Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

Summary

Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

Keywords

Heparin-induced thrombocytopenia - platelet factor 4 - GAGs - CD spectroscopy - antigenicity

^* Shared senior authorship.

• References

• 1 Schellekens H. The immunogenicity of therapeutic proteins. Discov Med 2010; 49: 560-564.
• 2 Schellekens H. Bioequivalence and the immunogenicity of biopharmaceuticals. Nat Rev Drug Discov 2002; 01: 457-462.
• 3 Moreland LW, McCabe DP, Caldwell 3rd JR. et al. Phase I/II trial of recombinant methionyl human tumor necrosis factor binding protein PEGylated dimer in patients with active refractory rheumatoid arthritis. J Rheumatol 2000; 27: 601-609.
• 4 Rau R, Sander O, van Riel P. et al. Intravenous human recombinant tumor necrosis factor receptor p55-Fc IgG1 fusion protein Ro 45–2081 (lenercept): a double blind, placebo controlled dose-finding study in rheumatoid arthritis. J Rheumatol 2003; 30: 680-690.
• 5 Eichler P, Lubenow N, Strobel U. et al. Antibodies against lepirudin are polyspecific and recognize epitopes on bivalirudin. Blood 2004; 103: 613-616.
• 6 Greinacher A, Lubenow N, Eichler P. Anaphylactic and anaphylactoid reactions associated with lepirudin in patients with heparin-induced thrombocytopenia. Circulation 2003; 108: 2062-2065.
• 7 Casadevall N, Nataf J, Viron B. et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 2002; 346: 469-475.
• 8 van Beers MMC, Sauerborn M, Gilli F. et al. Aggregated recombinant human interferon beta induces antibodies but no memory in immune tolerant transgenic mice. Pharm Res 2010; 27: 1812-1824.
• 9 Hemmer B, Stüve O, Kieseier B. et al. Immune response to immunotherapy: the role of neutralising antibodies to interferon beta in the treatment of multiple sclerosis. Lancet Neurol 2005; 04: 403-412.
• 10 Arepally GM, Ortel TL. Clinical practice. Heparin-induced thrombocytopenia. N Engl J Med 2006; 355: 809-817.
• 11 Wolpe SD, Cerami A. Macrophage inflammatory protein-1 and protein-2 – members of a novel superfamily of cytokines. Faseb J 1989; 03: 2565-2573.
• 12 Huang SS, Huang JS, Deuel TF. Proteoglycan carrier of human-platelet factor-4 – isolation and characterisation. J Biol Chem 1982; 257: 1546-1550.
• 13 Kaplan KL, Broekman MJ, Chernoff A. et al. Platelet alpha-granule proteins: studies on release and subcellular localisation. Blood 1979; 53: 604-618.
• 14 Rucinski B, Niewiarowski S, James P. et al. Antiheparin proteins secreted by human platelets. purification, characterisation, and radioimmunoassay. Blood 1979; 53: 47-62.
• 15 Deuel TF, Keim PS, Farmer M. et al. Amino-acid sequence of human platelet factor 4. PNAS 1977; 74: 2256-2258.
• 16 Greinacher A, Gopinadhan M, Günther J-U. et al. Close approximation of two platelet factor 4 tetramers by charge neutralisation forms the antigens recognized by HIT antibodies. Arterioscl Thromb Vasc Biol 2006; 26: 2386-2393.
• 17 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: 131-138.
• 18 Chong BH, Pitney WR, Castaldi PA. Heparin-induced thrombocytopenia – association of thrombotic complications with heparin-dependent Igg antibody that induces thromboxane synthesis and platelet-aggregation. Lancet 1982; 02: 1246-1249.
• 19 Amiral J, Bridey F, Dreyfus M. et al. Platelet factor-iv complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia. Thromb Haemost 1992; 68: 95-96.
• 20 Warkentin TE, Greinacher A. Heparin-induced thrombocytopenia: recognition, treatment, and prevention. Chest 2004; 126: 311s-337s.
• 21 Cines D, Kaywin P, Bina M. et al. Heparin associated thrombocytopenia. N Engl J Med 1980; 303: 788-795.
• 22 Chong BH, Fawaz I, Chesterman CN. et al. Heparin induced thrombocytopenia: mechanism of interaction of the heparin dependent antibody with platelets. Br J Haematol 1989; 73: 235-240.
• 23 Alban S, Krauel K, Greinacher A. Role of Sulfated Polysaccharides in the Pathogenesis of Heparin-Induced Thrombocytopenia. Heparin-Induced-Thrombocytopenia (Fundamental and Clinical Cardiology). 5. LLC: Boca Raton, USA: Taylor and Francis Group; 2013: 181-208.
• 24 Kelton JG, Smith J W, Warkentin TE. et al. Immunoglobulin-G from Patients with Heparin-Induced Thrombocytopenia Binds to a Complex of Heparin and Platelet Factor-4. Blood 1994; 83: 3232-3239.
• 25 Greinacher A, Alban S, Dummel V. et al. Characterisation of the structural requirements for a carbohydrate based anticoagulant with reduced risk of inducing the immunological type of heparin-associated thrombocytopenia. Thromb Haemost 1995; 74: 886-892.
• 26 Jaax ME, Krauel K, Marschall T. et al. Complex formation with nucleic acids and aptamers alters antigenic properties of platelet factor 4. Blood 2013; 122: 272-281.
• 27 Mikhailov D, Young HC, Linhardt RJ. et al. Heparin dodecasaccharide binding to platelet factor-4 and growth-related protein-alpha. J Biol Chem 1999; 274: 25317-25329.
• 28 Bohm G, Muhr R, Jaenicke R. Quantitative-analysis of protein far UV circular-dichroism spectra by neural networks. Protein Engin 1992; 05: 191-195.
• 29 Juhl D, Eichler P, Lubenow N. et al. 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: 420-426.
• 30 Sreerama N, Woody RW. Computation and Analysis of Protein Circular Dichroism Spectra. Methods in Enzymology 383. San Diego, CA: Elsevier Academic Press; 2004: 318-351.
• 31 Krauel K, Pötschke C, Weber C. et al. Platelet factor 4 binds to bacteria-inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia. Blood 2011; 117: 1370-1378.
• 32 Rosenthal MA, Rischin D, McArthur G. et al. Treatment with the novel anti-angiogenic agent PI-88 is associated with immune-mediated thrombocytopenia. Ann Oncol 2002; 13: 770-776.
• 33 Mayo KH, Barker S, Kuranda MJ. et al. Molten globule monomer to condensed dimer – role of disulfide bonds in platelet factor-iv folding and subunit association. Biochemistry 1992; 31: 12255-12265.
• 34 Villanueva GB, Allen N, Walz D. Circular-dichroism of platelet factor-4. Arch Biochem Biophys 1988; 261: 170-174.
• 35 Charles RS, Walz DA, Edwards BFP. The 3-dimensional structure of bovine platelet factor-4 at 3.0-A resolution. J Biol Chem 1989; 264: 2092-2099.
• 36 Gans PJ, Lyu PC, Manning MC. et al. The helix-coil transition in heterogeneous peptides with specific side-chain interactions: theory and comparison with CD spectral data. Biopolymers 1991; 31: 1605-1614.
• 37 Chin D-H, Woody RW, Rohl CA. et al. Circular dichroism spectra of short, fixed-nucleus alanine helices. PNAS 2002; 99: 15416-15421.
• 38 Leroux D, Canépa S, Viskov C. et al. Binding of heparin-dependent antibodies to PF4 modified by enoxaparin oligosaccharides: evaluation by surface plasmon resonance and serotonin release assay. J Thromb Haemost 2012; 10: 430-436.
• 39 Petitou M, Hérault JP, Bernat A. et al. Synthesis of thrombin-inhibiting heparin mimetics without side effects. Nature 1999; 398: 417-422.
• 40 Joglekar MV, Quintana Diez PM, Marcus S. et al. Disruption of PF4/H multi-molecular complex formation with a minimally anticoagulant heparin (ODSH). Thromb Haemost 2012; 107: 717-725.
• 41 Warkentin TE, Cook RJ, Marder VJ. et al. Anti-platelet factor 4/heparin antibodies in orthopedic surgery patients receiving antithrombotic prophylaxis with fondaparinux or enoxaparin. Blood 2005; 106: 3791-3796.
• 42 Warkentin TE, Pai M, Sheppard JI. et al. Fondaparinux treatment of acute heparin-induced thrombocytopenia confirmed by the serotonin-release assay: a 30-month, 16-patient case series. J Thromb Haemost 2011; 09: 2389-2396.
• 43 Sachais BS, Litvinov RI, Yarovoi SV. et al. Dynamic antibody-binding properties in the pathogenesis of HIT. Blood 2012; 120: 1137-1142.
• 44 Greinacher A, Michels I, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: the antibody is not heparin specific. Thromb Haemost 1992; 67: 545-549.
• 45 Krauel K, Fürll B, Warkentin TE. et al. Heparin-induced thrombocytopenia–therapeutic concentrations of danaparoid, unlike fondaparinux and direct thrombin inhibitors, inhibit formation of platelet factor 4–heparin complexes. J Thromb Haemost 2008; 06: 2160-2167.
• 46 Zhang X, Chen L, Bancroft DP. Crystal-structure of recombinant human platelet factor-4. Biochemistry 1994; 33: 8361-8366.
• 47 Chou K-C, Nemethy G, Rumsey S. et al. Interactions between an alpha-helix and a beta-sheet energetics of alpha-beta-packing in proteins. J Mol Bio 1985; 186: 591-609.
• 48 Ziporen L, Li ZQ, Park KS. et al. Defining an antigenic epitope on platelet factor 4 associated with heparin-induced thrombocytopenia. Blood 1998; 92: 3250-3259.
• 49 Li ZQ, Liu W, Park KS. et al. Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody. Blood 2002; 99: 1230-1236.
• 50 Arepally GM, Park KS, Kamei S. et al. Characterisation of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies. Blood 2000; 95: 1533-1540.
• 51 Cuker A, Rux AH, Hinds JL. et al. Novel diagnostic assays for heparin-induced thrombocytopenia. Blood 2012; 120: 267.
• 52 Suh JS, Aster RH, Visentin GP. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis recognize different epitopes on heparin: platelet factor 4. Blood 1998; 91: 916-922.