Charakterisierung von Plättchenfaktor 4-Polyanion Komplexen und korrespondierender Pathogener Antikörpern
Hämatologie, Onkologie
Zusammenfassung der Projektergebnisse
With this project, we have identified several important issues in heparin-induced thrombocytopenia (HIT). Firstly, we proved a new mechanism of autoimmunity caused by autoimmune antibodies. We established a two-step chromatography method to isolate human anti-platelet factor 4/Heparin antibodies (aPF4/H Abs). The aPF4/H Abs were then characterized by biophysical methods. We found that autoimmune group-3 antibodies mediate a new mechanism of autoimmunity. In particular, they bind to PF4/H complexes with a much higher binding force than others. Importantly, they have an ability to self-cluster PF4, forming antigenic complexes which allow binding of group-2 Abs. The resulting immunocomplexes induce massive platelet activation even in the absence of heparin. Antibody-mediated changes in endogenous proteins that trigger binding of otherwise non-pathogenic (or cofactor-dependent) antibodies may also be relevant in other antibody-mediated autoimmune disorders. Transferring the mechanism of the anti-PF4 autoimmune process to other autoimmune disorders may explain an unresolved issue regarding the pathogenicity of certain autoantibodies. In nearly all antibody-mediated autoimmune diseases, the antibody specificities found in severely affected patients are also found in asymptomatic individuals of the general population. It is an intriguing hypothesis that many of these antibodies are not pathogenic antibodies themselves. Rather, they can be recruited into the autoimmune process in the presence of small amounts of additional antibodies showing similar features as the group-3 Abs, i.e. they are able to change the structure of an endogenous protein thereby expressing an epitope, which allows the recruitment of otherwise nonpathogenic antibodies. Furthermore, both monoclonal and human clinically relevant aPF4/H Abs increase their binding forces to PF4 bound platelets compared with PF4 coated on the solid phase. Importantly, only group-3 Abs showed high binding forces to platelets without the addition of PF4. The finding allowed us to propose a platelet membrane-based expression of antigens for improvement of the specificity of HIT detection. Secondly, we identified a promising cell line that becomes promising materials for the development cell-based assays for better detection of the clinically relevant aPF4/H antibodies. These aPF4/H Abs bind firmly on PF4-bound breast cancer cell line (MDA-MB-231). Importantly, Abs bound cells could be detected by different methods such as PF4-ELISA, flow cytometry, and confocal microscopy. This identified cell line became an advantage in HIT diagnostics as it can replace the use of platelets that are isolated from fresh human blood. Thirdly, we found several conditions that allow to improve the current detection of HIT Abs. The non-pathogenic antibodies only slightly reduced binding in the PF4 ELISA test while pathogenic antibodies showed a strong reduction of binding at 500 mM NaCl. The buffer condition (i.e. 50 mM salt, pH6.0) allows better differentiation between clinically relevant platelet activating aPF4/P antibodies and clinically non-relevant non-activating ones in PF4 ELISA. We recognize that PF4 could form aggregation at several storage conditions. This is a caution for in vivo tests as aggregated PF4 can alter the results. Our results on specific physical characteristics of clinically relevant, plateletactivating aPF4/H Abs provide the basis for better diagnostic approaches for today’s most frequent adverse drug effects affecting blood cells. We also identified a certain concentration ratio between PF4 and fondaparinux that could form sufficiently large and stable complexes that stabilize the binding of the KKO and enhance platelet aggregation and activation. We provided a model to explain controversial observations of fondaparinux-induced thrombocytopenia in patients as each contains a dissimilar PF4:fondaparinux concentration ratio. Lastly, we characterized newly developed humanized aPF4/H antibodies that helped to commercialize the second HIT-mimick aPF4/H Abs which is important for many research groups that do not have access to human materials. We have successfully characterized the humanized HIT-like antibodies (5B9 and 1E12) that showed similar binding characteristics of HIT and autoimmune HIT aPF4/H Abs group-3), respectively. The 5B9 antibody is now commercially available by Stago company while the 1E12 is used in several Labs as an autoimmune-mimick aPF4/H Abs. Characterization of PF4 mutants by biophysical methods allowed us to identify two PF4 mutants (L8A and Delta 1-8a.a.) that allowed us to better differentiate between HIT and non-HIT sera. Even though we could not prove that PF4 bind to vWF due to technical limitation, other colleagues have in parallel proved our hypothesis.
Projektbezogene Publikationen (Auswahl)
- Anti-platelet factor 4/polyanion antibodies mediate a new mechanism of autoimmunity. Nature Communications. (2017) 8
Nguyen T.H., Medvedev N., Delcea M., Greinacher A.
(Siehe online unter https://doi.org/10.1038/ncomms14945) - Effect of pH and ionic strength on the binding strength of anti- PF4/polyanion antibodies. Eur Biophys J. (2017)
Nguyen T.H., Greinacher A.
(Siehe online unter https://doi.org/10.1007/s00249-017-1240-8) - Platelet factor 4/heparin complexes present their epitopes differently on a solid phase system than on the platelet surface. Blood (2017)
Nguyen T.H., Greinacher A.
(Siehe online unter https://doi.org/10.1182/blood-2017-02-769786) - Distinct Binding Characteristics of Pathogenic Anti-Platelet Factor-4/Polyanion Antibodies to Antigens Coated on Different Substrates: A Perspective on Clinical Application, ACS Nano, (2018)
Nguyen T.H., Greinacher A.
(Siehe online unter https://doi.org/10.1021/acsnano.8b04487) - Not Only Heparin but Also Antibody Induces Thrombocytopenia. Book title: “Thrombocytopenia.” Edited by Pankaj Abrol, ISBN 978-953-51-3876-1, IntechOpen (2018)
Nguyen T.H.
(Siehe online unter https://doi.org/10.5772/intechopen.73576) - Characterization of the Interaction between Platelet Factor 4 and Homogeneous Synthetic Low Molecular Weight Heparins, J Thromb Haemost. (2019)
Nguyen T.H., Xu Y., Brandt S., Mandelkow M., Raschke R., Strobel U, Delcea M., Zhou W., Liu J., and Greinacher A.
(Siehe online unter https://doi.org/10.1111/jth.14657) - Heparin-induced thrombocytopenia Assay, United States Patent, WO 2019/202395
Greinacher A., Nguyen T.H.
- Reactivity of platelet‐activating and nonplatelet‐activating anti‐PF4/heparin antibodies in enzyme immunosorbent assays under different conditions, J. Thrombosis and Haemostasis, 17 (2019) 1113-1119
Nguyen T.H., Wesche J., Raschke R., Strobel U., Bui C.C., Delcea M. and Greinacher A.
(Siehe online unter https://doi.org/10.1111/jth.14455) - Characterization of new Monoclonal PF4-specific Antibodies as useful Tools for Studies on Typical and Autoimmune HIT, Thrombosis and Haemostasis (2020)
Vayne C., Nguyen T.H., Rollin J., Charuel N., Poupon A., Pouplard C., Normann N., Gruel Y., and Greinacher A.
(Siehe online unter https://doi.org/10.1055/s-0040-1717078) - Physicochemical Characteristics of Platelet Factor 4 under Various Conditions are Relevant for Heparin-Induced Thrombocytopenia Testing, J. Physical Chemistry B (2020)
Bui V.C., Gebicka P., Hippe H., Raschke R., Nguyen T.L., Greinacher A., Nguyen T.H.
(Siehe online unter https://doi.org/10.1021/acs.jpcb.9b11695) - Effect of HIT Components on the Development of Breast Cancer Cells, Life (2021)
Chen L.Y., Apte G., Lindenbauer A., and Nguyen T.H.
(Siehe online unter https://doi.org/10.3390/life11080832) - Label-Free Detection and Characterization of Heparin-induced Thrombocytopenia-like Antibodies, ACS Omega (2021)
Khan ZN, Chen L.Y., Lindenbauer A., Pliquett U., Rothe H., Nguyen T.H.
(Siehe online unter https://doi.org/10.1021/acsomega.1c02496) - Method for detection of heparin-induced thrombocytopenia antibodies, European Patent, Appl. Nr: EP21180857.1 (06/2021)
Nguyen T.H., Chen L.Y., and Apte G.
- Breast cancer cell-based ELISA: A potential material for better detection of heparin-induced thrombocytopenia antibodies, Journal of Materials Chemistry B (2022)
Chen L.Y., Schirmer U., Widder M., Gruel Y., Rollin J., Zipfel P., and Nguyen T.H.
(Siehe online unter https://doi.org/10.1039/d2tb01228f) - When will fondaparinux induce thrombocytopenia? Bioconjugate Chemistry (2022)
Chen LY, Khan ZN, Lindenbauer A, and Nguyen T.H.
(Siehe online unter https://doi.org/10.1021/acs.bioconjchem.2c00316)
