Biophysical tools to assess the interaction of PF4 with polyanions

Mihaela Delcea; Andreas Greinacher

doi:10.1160/TH16-04-0258

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2016; 116(05): 783-791
DOI: 10.1160/TH16-04-0258

Theme Issue Article

Schattauer GmbH

Biophysical tools to assess the interaction of PF4 with polyanions

Authors

Mihaela Delcea

¹Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany

²ZIK HIKE - Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany

³DZHK (German Centre for Cardiovascular Research), partner site Greifswald, Germany
Andreas Greinacher

¹Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany

Abstract

Summary

The antigen in heparin-induced thrombocytopenia (HIT) is expressed on platelet factor 4 (PF4) when PF4 complexes with polyanions. In recent years, biophysical tools (e. g. circular dichroism spectroscopy, atomic force microscopy, isothermal titration calorimetry, x-ray crystallography, electron microscopy) have gained an important role to complement immunological and functional assays for better understanding the interaction of heparin with PF4. This allowed identification of those features that make PF4 immunogenic (e. g. a certain conformational change induced by the polyanion, a threshold energy of the complexes, the existence of multimeric complexes, a certain number of bonds formed by PF4 with the polyanion) and to characterize the morphology and thermal stability of complexes formed by the protein with polyanions. These findings and methods can now be applied to test new drugs for their potential to induce the HIT-like adverse drug effect by preclinical in vitro testing. The methods and techniques applied to characterize the antigen in HIT may also be helpful to better understand the mechanisms underlying other antibody-mediated disorders in thrombosis and hemostasis (e. g. acquired hemophilia, thrombotic thrombocytopenic purpura). Furthermore, understanding the mechanisms making the endogenous protein PF4 immunogenic may help to understand the mechanisms underlying other autoimmune disorders.

Keywords

Platelet factor 4 - heparin - conformational changes - heparin-induced thrombocytopenia - biophysics of PF4/polyanion complexes

Full Text

References

References
1 McGowan KE, Makari J, Diamantouros A. et al. Reducing the hospital burden of heparin-induced thrombocytopenia: impact of an avoid-heparin program. Blood 2016; 127: 1954-1959.
2 Greinacher A, Warkentin TE. Risk of heparin-induced thrombocytopenia in patients receiving thromboprophylaxis. Exp Rev Hematol 2008; 01: 75-85.
3 Kelton JG, Sheridan D, Santos A. et al. Heparin-Induced Thrombocytopenia - Laboratory Studies. Blood 1988; 72: 925-930.
4 Tutwiler V, Madeeva D, Ahn HS. et al. Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia. Blood 2016; 127: 464-472.
5 Greinacher A, Gopinadhan M, Gunther JU. et al. Close approximation of two platelet factor 4 tetramers by charge neutralisation forms the antigens recognized by HIT antibodies. Arterioscler Thromb Vasc Biol 2006; 05: 2386-2393.
6 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: 2442-2449.
7 Villanueva GB, Allen N, Walz D. Circular-Dichroism of Platelet Factor-4. Arch Biochem Biophys 1988; 261: 170-174.
8 Brandt S, Krauel K, Gottschalk KE. et al. Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity. Thromb Haemost 2014; 112: 53-64.
9 Jaax ME, Krauel K, Marschall T. et al. Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4. Blood 2013; 122: 272-281.
10 Brandt S, Krauel K, Jaax M. et al. Polyphosphates form antigenic complexes with platelet factor 4 (PF4) and enhance PF4-binding to bacteria. Thromb Haemost 2015; 114: 1189-1198.
11 Delcea M, Kreimann M, Greinacher A. Thermal stability of platelet factor 4 and its complexes with heparins studied by differential scanning calorimetry. J Thromb Haemost 2015; 13: 213 (Abstract)
12 Mayo KH, Chen MJ. Human platelet factor 4 monomer-dimer-tetramer equilibria investigated by 1H NMR spectroscopy. Biochemistry 1989; 28: 9469-9478.
13 Mayo KH, Barker S, Kuranda MJ. et al. Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association. Biochemistry 1992; 31: 12255-12265.
14 Mayo KH, Ilyina E, Roongta V. et al. Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding. Biochem J 1995; 312: 357-365.
15 Mikhailov D, Young HC, Linhardt RJ. et al. Heparin dodecasaccharide binding to platelet factor-4 and growth-related protein-alpha. Induction of a partially folded state and implications for heparin-induced thrombocytopenia. J Biol Chem 1999; 274: 25317-25329.
16 Block S, Greinacher A, Helm CA. et al. Characterisation of bonds formed between platelet factor 4 and negatively charged drugs using single molecule force spectroscopy. Soft Matter 2014; 10: 2775-2784.
17 Nguyen TH, Greinacher A, Delcea M. Quantitative description of thermody-namic and kinetic properties of the platelet factor 4/heparin bonds. Nanoscale 2015; 07: 10130-10139.
18 Sachais BS, Litvinov RI, Yarovoi SV. et al. Dynamic antibody-binding properties in the pathogenesis of HIT. Blood 2012; 120: 1137-1142.
19 Litvinov RI, Yarovoi SV, Rauova L. et al. Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4. J Biol Chem 2013; 288: 33060-33070.
20 Zhang X, Chen L, Bancroft DP. et al. Crystal structure of recombinant human platelet factor 4. Biochemistry 1994; 33: 8361-8366.
21 Cai Z, Yarovoi SV, Zhu Z. et al. Atomic description of the immune complex involved in heparin-induced thrombocytopenia. Nat Commun 2015; 06: 8277.
22 Suvarna S, Espinasse B, Qi R. et al. Determinants of PF4/heparin immunogenic-ity. Blood 2007; 110: 4253-4260.
23 Rauova L, Poncz M, McKenzie SE. et al. Ultralarge complexes of PF4 and hepa-rin are central to the pathogenesis of heparin-induced thrombocytopenia. Blood 2005; 105: 131-138.
24 Medvedev N, Palankar R, Krauel K. et al. Micropatterned array to assess the interaction of single platelets with platelet factor 4-heparin-IgG complexes. Thromb Haemostasis 2014; 111: 862-872.
25 Greinacher A, Potzsch B, Amiral J. et al. Heparin-associated thrombocytopenia: isolation of the antibody and characterisation of a multimolecular PF4-heparin complex as the major antigen. Thromb Haemost 1994; 71: 247-251.
26 Sachais BS, Rux AH, Cines DB. et al. Rational design and characterisation of platelet factor 4 antagonists for the study of heparin-induced thrombocytope-nia. Blood 2012; 119: 5955-5962.
27 Li ZQ, Liu WY, 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.
28 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.
29 Kelton JG, Smith JW, 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.
30 Greinacher A, Alban S, Dummel V. et al. Characterisation of the structural requirements for a carbohydrate based anticoagulant with a reduced risk of inducing the immunological type of heparin-associated thrombocytopenia. Thromb Haemost 1995; 74: 886-892.
31 Visentin GP, Moghaddam M, Beery SE. et al. Heparin is not required for detection of antibodies associated with heparin-induced thrombocytopenia/throm-bosis. J Lab Clin Med 2001; 138: 22-31.
32 Amiral J, Bridey F, Wolf M. et al. Antibodies to Macromolecular Platelet Factor 4-Heparin Complexes in Heparin-Induced Thrombocytopenia - a Study of 44 Cases. Thromb Haemost 1995; 73: 21-28.
33 Bock PE, Luscombe M, Marshall SE. et al. The multiple complexes formed by the interaction of platelet factor 4 with heparin. Biochem J 1980; 191: 769-776.
34 Stuckey JA, St Charles R, Edwards BF. A model of the platelet factor 4 complex with heparin. Proteins 1992; 14: 277-287.
35 Cowan SW, Bakshi EN, Machin KJ. et al. Binding of heparin to human platelet factor 4. The Biochemical journal 1986; 234: 485-488.
36 Ibel K, Poland GA, Baldwin JP. et al. Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering. Biochim Biophys Acta 1986; 870: 58-63.
37 Savi P, Chong BH, Greinacher A. et al. Effect of fondaparinux on platelet activation in the presence of heparin-dependent antibodies: a blinded comparative multicenter study with unfractionated heparin. Blood 2005; 105: 139-144.
38 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.
39 Greinacher A, Amiral J, Dummel V. et al. Laboratory diagnosis of heparin-as-sociated thrombocytopenia and comparison of platelet aggregation test, hepa-rin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay. Transfusion 1994; 34: 381-385.
40 Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced throm-bocytopenia. Blood 1986; 67: 27-30.
41 Padmanabhan A, Jones CG, Bougie DW. et al. Heparin-independent, PF4-de-pendent binding of HIT antibodies to platelets: implications for HIT pathogen-esis. Blood 2015; 125: 155-161.
42 Nazi I, Arnold DM, Warkentin TE. et al. Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced throm-bocytopenia. J Thromb Haemost 2015; 1900-1907.
43 Warkentin TE, Basciano PA, Knopman J. et al. Spontaneous heparin-induced thrombocytopenia syndrome: 2 new cases and a proposal for defining this disorder. Blood 2014; 123: 3651-3654.
44 Greinacher A. Me or not me? The danger of spontaneity. Blood 2014; 123: 3536-3538.