@phdthesis{Kreimann2015,
  author    = {Martin Kreimann},
  title     = {Characterization of complexes between platelet factor 4 and heparin},
  journal    = {Charakterisierung von Komplexen zwischen den Pl{\"a}ttchenfaktor 4 und Heparin},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-002127-7},
  year      = {2015},
  abstract  = {Heparin is an anticoagulant drug. It is important in the treatment of deep vein thrombosis,pulmonary embolism and during surgeries. Heparin-induced thrombocytopenia (HIT) is a severe adverse reaction caused by the formation of ultralarge complexes of platelet factor 4 (PF4) with unfractionated heparin (UFH). It can lead to limb loss or fatal events like stroke, myocardial infarction or pulmonary embolism. HIT has an incidence of about 3\% in patients receiving anticoagulative heparin treatment. PF4 is a tetrameric protein, released from the α-granules of platelets upon activation. PF4 is known to form antigenic complexes with UFH accompanied by structural changes of PF4. In this thesis, the size and size distribution of PF4 and PF4/heparin complexes were analyzed using asymmetrical flow field-flow-fractionation (AF4), photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). PF4 tends to form auto-aggregates and to adsorb to different surfaces, including regenerated cellulose, polyethersulfone, quartz and glass. The aggregates are less pronounced in solutions at isotonic NaCl concentration. Arginine and Tween 20 were identified as possible ingredients to hinder the auto-aggregation of PF4. Also, it is shown by combining circular dichroism (CD) spectroscopy, atomic force microscopy (AFM) and isothermal titration calorimetry (ITC) with UFH and defined chain length (16-, 8-, 6-, 5-mer) heparins that structural changes (i.e., increase in \β-sheets) alone are not sufficient to induce antigenicity. While UFH, 16-, 8-, and 6-mer heparins all induced an increase in the antiparallel \β-sheet content to > 30\% (as determined by CD spectroscopy), complex antigenicity as measured by anti-PF4/heparin antibody binding in an enzyme-linked immunosorbent assay (EIA) was only induced by UFH and 16-mer heparin. Fondaparinux (5-mer heparin), which forms in vitro non-antigenic complexes with PF4, did not induce structural changes of PF4. Interestingly, the structural changes induced by antigenic UFH and 16-mer heparin but not by non-antigenic shorter heparins were reversible at higher heparin concentrations. Furthermore, the complexes formed by PF4 with longer heparins were larger than those formed with shorter heparins as shown by atomic force microscopy (AFM). UFH, HO16 and HO08 are able to form ultralarge multimolecular complexes with PF4. ITC data indicated strong electrostatic interactions and energetically unfavorable conformational changes of PF4 with longer heparins, while for the short heparins, favorable conformational changes in the structure of PF4 are induced. This explains the reversibility of the structural changes seen for UFH and HO16 upon addition of an over-saturating amount of heparin. Finally, using differential scanning calorimetry (DSC) the thermal stability of PF4 and PF4/heparin complexes was assessed. Despite its tendency to form auto-aggregates, PF4 is a heat-stable protein. This stability is, length dependently, even increased in complex with heparins. This work shows important differences in the binding between PF4 and heparins of different chain length and might be relevant for the understanding of other biological functions of heparins (e.g., involvement in allergic and inflammatory reactions).},
  language  = {en}
}