Heparin-induced thrombocytopenia (HIT)

When receiving anticoagulant heparin (H), up to ~3% patients develop anti-platelet factor 4 (PF4)/H antibodies. These antibodies bind to PF4/H complexes followed by bridging and activating platelets, thereby forming platelet aggregation and activation. A subset of these antibodies selects their own antigens(https://www.nature.com/articles/ncomms14945), binds  strongly to platelets, and activates them (https://www.ncbi.nlm.nih.gov/pubmed/30540167).

This side effect is called heparin-induced thrombocytopenia (HIT) which can result in life-threatening. It has been shown that long heparins induce more frequent HIT than short ones (https://pubs.rsc.org/en/Content/ArticleLanding/NR/2015/C5NR02132D#!divAbstract), while synthetic heparins increase binding to PF4 (https://www.ncbi.nlm.nih.gov/pubmed/31573759).

Heparin-induced thrombocytopenia (HIT)

Heparins can form ultra large complexes with PF4, allowing binding of several antibodies. These antibodies can bridge between platelets and activate them.

The NFG focuses on further understanding HIT complications, including studies on antibodies, heparins, PF4s, platelets and involving cells. We not only answer fundamental questions on HIT but also develop methods for better detection of HIT antibodies.

  • Projekte
    • Methodologies

      Single-molecule force spectroscopy based atomic force spectroscopy, Fast Protein Liquid Chromatography (FPLC), Quartz Crystal Microbalance (QCM), and Electrochemical Impedance Spectroscopy (EIS), Two-Photon Polymerization (2PP), Scanning Electron Microscopy (SEM), Confocal Laser Microscopy (CLMS), Enzyme immunoassays, Dynamic Light Scattering (DLS), Circular dichroism (CD) spectroscopy, Surface Plasmon Resonance (SPR), Enzyme Immunoassays (EIA), Flow Cytometry (FC), Microfluidics, etc.

    • Synthesis and characterization of magnetic nanoparticles

      Platelets are transfused to prevent bleeding or to treat bacterial infection in many patients. To distinguish transfused platelets from the patient’s, own after transfusions, platelets are labeled with magnetic nanoparticles. Platelet labeling efficiency is enhanced when particles are conjugated with proteins and binding pathways of particles during platelet labeling have been determined (ACS Appl. Mater. Interfaces 2018, 10, 34, 28314-28321). However, the large variety of binding forces between particle and platelet  and the aggregation of particles were still observed. This limitation may be due to the original characteristics of magnetic nanoparticles such as their shape/size, morphology and also the type of proteins conjugated on the surface of the particles.

      Development of magnetic nanoparticles.
      Iron oxide core of different shapes such as sphere and cubic shapes are synthesized and conjugated with protein.

      To further improve platelet labeling, we cooperate with Dr. Jörg Schemberg to synthesize magnetic nanoparticles with different shapes/morphologies and to narrow the size distribution of the nanoparticles. We conjugate magnetic nanoparticles with proteins and characterize them when they interact with human platelets.

    • HIT antibodies – Cancer cells

      Many cancer patients are treated with heparins. Patients with heparin treatment may develop HIT antibodies. However, little is known about the role of HIT antibodies in cancer patients. We aim to investigate the interaction of HIT antibodies with cancer cells. Insights into the binding of HIT antibodies to cancer cells may help us to better manage HIT in cancer patients.

      Platelets bound to breast cancer cells (left)
      cultured on a petri dish and (right) in fluid phase.

    • Controlling platelet-surface activation

      Platelets can adhere to almost all nonphysiological surfaces and are quickly activated. Modulation of platelet-surface activation is important for many medical applications. We recently found that the degree of platelet-surface activation could be reduced by coating surfaces with collagen-G (Scientific Reports, 6(1), 25402, July 2016) or when they are seed on laminin coated nano-groove patterns. However, weak activation is still observed on all investigated surfaces. To further reduce platelet-surface activation, we aim to produce nanopatterns and nanoscaftfolds of different shapes and sizes using various types of polymers and modify them before seeding platelets on.

      Controlling platelet-surface activation.

      Nanostructured surfaces are fabricated and modified with different materials before seeding platelets on. 

    • Detection of HIT antibodies

      Current enzyme immunoassays based optical detection are widely used due to their high sensitivity and fast turnover rate but the accuracy of these techniques is just ~50%. As HIT antibodies are the central problem that leads to platelet aggregation and activation, a technique for better detection of these antibodies is urgently needed. A false result may conduct treatment for patients without containing pathogenic HIT antibodies that can result in serious consequences such as venous limb gangrene or fatal hemorrhage. Recently, we found that HIT antibodies bind strongly to platelet membrane (Blood (2017) 129 (26): 3498–3501), (ACS Nano. 2018 Dec 26;12(12):12030-12041) and binding characteristics of HIT antibodies depend on their surrounding environment (J Thromb Haemost. 2019 Jul;17(7):1113-1119), (J. Phys. Chem. B 2020, 124, 8, 1438-1443). Based on these findings, we aim in two directions to better detect pathogenic HIT antibodies:

      Development of an electrical biosensor

      PF4/Heparin complexes are covalently immobilized on Au-sensor. HIT antibodies are added into the chamber for interaction with PF4/H complexes. Binding of HIT antibodies will be detected by impedance signals.

      Biosensor for detection of HIT antibodies
      Example of impedance signals show for baseline (blue), 1st (red), 2nd (green) and 3rd (violet) concentration added.

      Improvement of current PF4 enzyme immunoassays (EIA)

      PF4/heparin complexes are immobilized on a 96-well plate to allow binding of anti-PF4/heparin antibodies. They are subsequently detected by a secondary goat anti-human IgG antibody using a chromogenic substrate that yields a visible color change, indicating their binding to the coated PF4/heparin complexes. The optical density (OD) of the sample was measured at wavelength 450 nm. 

      PF4 EIA. (left) Schematic model for the detection of HIT antibodies in EIA. (right) Current PF4 EIA provides an overlap OD signal (red area) of ~50% between HIT (red line) and non-HIT (black line) antibodies. 

  • Publikationen

    Bui V.C., Nguyen T.H., Direct monitoring of drug-induced mechanical response of individual cells by atomic force microscopy, J. Molecular Recognition (2020), doi: 10.1002/jmr.2847.

    Bui V.C., Gebicka P., Hippe H., Raschke R., Nguyen T.L., Greinacher A., Nguyen T.H.*, Physicochemical Characteristics of Platelet Factor 4 under Various Conditions are Relevant for Heparin-Induced Thrombocytopenia Testing, J. Physical Chemistry (2020), doi.org/10.1021/acs.jpcb.9b11695.

    Nguyen T.H.*, Xu Y., Brandt S., Mandelkow M., Raschke R., Strobel U, Delcea M., Zhou W., Liu J., and Greinacher A.*, Characterization of the Interaction between Platelet Factor 4 and Homogeneous Synthetic Low Molecular Weight Heparins, J Thromb Haemost. (2019) Oct 1. doi: 10.1111/jth.14657.

    Nguyen T.H.*, Wesche J., Raschke R., Strobel U., Bui V.C., Delcea M. and Greinacher A.*, Reactivity of platelet-activating and nonplatelet-activating anti-PF4/heparin antibodies in enzyme immunosorbent assays under different conditions, J. Thromb Haemost, 17 (2019) 1113-1119.

    Nguyen T.H.*, Greinacher A.*, Distinct Binding Characteristics of Pathogenic Anti-Platelet Factor-4/Polyanion Antibodies to Antigens Coated on Different Substrates: A Perspective on Clinical Application, ACS Nano, 12(12), 12030-12041 (2018).

    Nguyen T.H.*, Schuster N., Greinacher A., Aurich K.*, Uptake pathways of protein-coated magnetic nanoparticles in platelets, ACS Applied Materials & Interfaces, 10 (34), 28314-21, (2018).

    Bui V.C., Nguyen T.H.*, The Role of Single-Molecule Force Spectroscopy in Unraveling Typical and Autoimmune Heparin-induced Thrombocytopenia” Int. J. Mol. Sci., 19, 1054 (2018).doi: 10.3390/ijms19041054.

    Bui V.C., Nguyen T.H.*, DNA aggregation induced by Mg2+ ions under different conditions, J Mol Recognit, (2018). DOI: 10.1002/jmr.2721.

    Bui V.C., Nguyen T.H.*, Biophysical Characteristics of Hematopoietic Cells during Division, Exp Cell Res., 367(2),132-136 (2018).

    Nguyen T.H.*, Medvedev N., Delcea M., Greinacher A.*, Anti-platelet factor 4/polyanion antibodies mediate a new mechanism of autoimmunity, Nature Communications 8:14945 (2017).

    Nguyen T.H.*, Greinacher A.*, Platelet factor 4/Heparin complexes present differently in the purified system and on the platelet surface, Blood, (2017).

    Nguyen T.H.*, Greinacher A., Effect of pH and Ionic Strength on the Binding Strength of PF4/Polyanion Antibodies, Eur Biophys J. (2017).

    Bui V.C., Nguyen T.H.*, Insights into the interaction of CD4 with anti-CD4 antibodies, Immunobiology, 222(2), 148 (2017).

    Nguyen T.H.*, Single Molecule Force Spectroscopy Applied to Heparin-Induced Thrombocytopenia, J Mol Recognit, 2585 (2017).

    Henrich F., Fell D., Truszkowska D., Weirich M., Anyfantakis M., Nguyen T.H., M. Wagner, Auernhammer G.K.* and Butt H.J., Influence of surfactants in forced dynamic dewetting, Soft Matter 12(37), 7782 (2016).

    Nguyen T.H.*, Palankar R., Bui V.C., Medvedev N., Greinacher A.*, and Delcea M.*. Rupture Forces among Human Blood Platelets at different Degrees of Activation, Scientific Reports 6,1- 12 (2016)

    Bui V.C., Nguyen T.H.*, The role of CD4 on mechanical properties of live cell membrane, J. Biomed. Mater. Res. A., 104(1), 239-44 (2016).

    Nguyen T.H.*, Greinacher A., Delcea M.*, Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds, Nanoscale 7, 10130-9 (2015).

    Lee S. M., Nguyen T.H., Na K., Cho I.J., Woo D.H., Oh J.E., Lee C.J., Yoon E.S.*, Nanomechanical measurement of astrocyte stiffness correlated with cytoskeletal maturation, J. Biomed. Mater. Res. A., 103(1) 365-370 (2014).

    Eibach T.F., Fell D., Nguyen T.H., Butt H.J., Auernhammer G. K.*, Measuring contact angle and meniscus shape with a reflected laser beam, Rev. Sci. Instrum., 85, 13703-11 (2014).

    Cui J.X., Nguyen T.H., Ceolin M., Berger R., Azzaroni O., del Campo A.*, Phototunable response in caged polymer brushes, Macromolecules, 45(7), 3213-3220 (2012).

    Nguyen T.H., Steinbock L.J., Butt H.J., Helm M., Berger R.*, Measuring single small molecule binding via rupture forces of a split aptamer, J. Am. Chem. Soc., 133, 2025-2027 (2011).

    Nguyen T.H., Lee S.M., Na K., Yang S., Kim J., Yoon E.S.*, An enhanced measurement of dsDNA elasticity using AFM, Nanotechnology, 21, 1-7 (2010).

    Nguyen T.H., Kim Y.U., Choi S.S*, Investigation of structural transition of dsDNA on various substrates studied by atomic force microscopy, J. Nanosci. Nanotech. 9, 2162-2168 (2009).

    Nguyen T.H., Bui V.C., Kim Y.N., Choi S.S.*, Tip functionalization by biotin linkers: a technique for DNA elasticity measurement studied by atomic force microscopy, J. Bionanosci., 3, 1-6 (2009).

    Nguyen T.H., Bui V.C., Choi S.S. *, Manipulation of Single Molecule: DNA Physical Characteristic Observation Studied with Atomic Force Microscopy, Advances in Natural Sciences, 9, 225-232 (2008).

    Nguyen T.H., Choi S.S., Kim Y.U., Kim D.W.*, Study on the binding force between λ-DNA and various types of substrates, J. Kor. Phys. Soc., 50, 1942-1946 (2007).

    Luu D.H.*, Nguyen T.H., Study on Epimerization of the (17-a)- Ethynylestradiol. J. Chem. 5, 72- 75 (2001).

    Nguyen X.D., Nguyen V.N.*, Tran T.M.L., Le V.H., Nguyen T.H., Nguyen T.T., The use of gracilaria biomass for removal of heavy metals from aqueous solutions, J. Chem. 3, 89-91 (2001).

  • Mitarbeiter

    Members

    Dr. Thi-Huong Nguyen, Group Leader
    Tel. +49 (0)3606 671 600
    Fax: +49 (0)3606 671 200
    Email: thi-huong.nguyennoSpam@heiligenstadt.de

    Download CV

    Mr. Gurunath Apte, PhD student
    Start: 02.09.2019
    He got his Master's degree from Martin Luther Universität Halle-Wittenberg & Anhalt University of Applied Sciences in June 2019. His master thesis focused on “Dip Coating process to create polymeric films for skin tissue regeneration”. 
    Reference: www.meta.org/papers/effect-of-different-crosslinking-strategies-on/31531939
    His PhD study at IBA: ‘Monitoring of platelet-surface activation by nanopatterning’.

    Tel. +49 (0)3606 671 600
    Email: Gurunath.AptenoSpam@iba-heiligenstadt.de

    Ms. Nida Khan, PhD student
    Start: 01.10.2019
    She got her Master’s degree at Gautam Buddha University, India in August 2019. Her master thesis focuses on “Cloning of Secreted modular calcium-binding protein 1 promoter”. 
    Her PhD thesis at IBA: ‘Development of a biosensor for the detection of pathogenic HIT antibodies’.

    Tel. +49 (0)3606 671 615
    Email: Nida.KhannoSpam@iba-heiligenstadt.de

    Mr. Li-Yu Chen, PhD student
    Start: 01.11.2019
    He got his Master's degree at University Jena, Germany in March 2019. His master thesis focused on the immunological reaction between staphylococcal protein PurA and human dectin-1 and dectin-2 receptors. 
    His PhD study at IBA: ‘Characterization of new materials for improvement of ELISA in detection of HIT antibodies’.

    Tel. +49 (0)3606 671 630
    Email: Li-Yu.ChennoSpam@iba-heiligenstadt.de

    Mrs. Annerose Lindenbauer, Technician
    Start: 01.07.2019
    Tel. +49 (0)3606 671 830
    Email: Annerose.LindenbauernoSpam@iba-heiligenstadt.de