Refine
Document Type
- Article (3)
- Doctoral Thesis (1)
Language
- English (4)
Has Fulltext
- yes (4)
Is part of the Bibliography
- no (4)
Keywords
- - (1)
- Biochemie (1)
- Integrin αIIbβ3 (1)
- detergents (1)
- integrin αIIbβ3 (1)
- lipids (1)
- liposomes (1)
- reconstitution (1)
Institute
Publisher
- MDPI (1)
Magnetic nanoparticles have a broad spectrum of biomedical applications including cell separation, diagnostics and therapy. One key issue is little explored: how do the engineered nanoparticles interact with blood components after injection? The formation of bioconjugates in the bloodstream and subsequent reactions are potentially toxic due to the ability to induce an immune response. The understanding of the underlying processes is of major relevance to design not only efficient, but also safe nanoparticles for e.g.
targeted drug delivery applications. In this study, we report on maghemite nanoparticles functionalized with citrate-, dextran- and polyethylene glycol coatings and their interaction with the clotting protein fibrinogen. Further, we investigate using biophysical tools (e.g. dynamic light scattering, circular dichroism spectroscopy and quartz crystal microbalance) the interaction of the magnetic nanoparticles–fibrinogen bioconjugates with artificial cell membranes as a model system for blood platelets. We found that fibrinogen corona formation provides colloidal stability to maghemite nanoparticles. In addition, bioconjugates of fibrinogen with dextran- and citrate-coated NPs interact with integrin-containing lipid bilayer, especially upon treatment with divalent ions, whereas PEG-coating reveals minor interaction. Our study at the interface of protein-conjugated nanoparticles and artificial cell membranes is essential for engineering safe nanoparticles for drug delivery applications.
Blood platelets are primary major players in the coagulation cascade, that act upon damage in blood vessels at the subendothelial surface. During this process, platelets change their shape, release granules and aggregate by cross-linking of integrin αIIbβ3 via fibrinogen. The heterodimeric transmembrane receptor integrin αIIbβ3 is highly expressed on platelets and its regulation is bidirectional. Inside-out signaling leads to increased affinity for ligands due to dramatic rearrangements in the integrin conformation changing from an inactive bent conformation to an extended, high-affinity conformation. The swing-out motion of the integrin head domain enables binding of ligands, e.g. fibrinogen, resulting in outside-in signaling guiding kinase activation, shape change, platelet aggregation and spreading, subsequently.
Agonists (e.g. thrombin) and other triggers (e.g. shear stress) promote the activity of platelets, making the study of specific proteins delicate. Therefore, this PhD thesis describes a biomimetic system used to study αIIbβ3 membrane receptors. Integrin αIIbβ3 was successfully reconstituted into liposomes and characterized by biophysical and molecular biological methods (e.g. dynamic light scattering, transmission electron microscopy, circular dichroism spectroscopy and flow cytometry). The fusion of liposomes to a solid substrate allows the analysis of potential activation triggers and interaction partners concerning their role in integrin αIIbβ3 activation in a lipid bilayer. Among others, quartz-crystal microbalance measurements show that divalent ions and clinically relevant drugs (e.g. unfractionated heparin and quinine), known to be involved in immune thrombocytopenia (ITP), are certainly candidates which induce integrin activation and minor changes in protein secondary structure. In addition, protein corona formation during contact of nanoparticles with blood components, such as fibrinogen, as well as their interaction with artificial platelet model membranes containing integrins were studied. Moreover, lipid environment can be strongly controlled as integrin activation is dependent on the ratio of liquid-ordered and disordered phases within the membrane. Eventually, by exclusion of disturbances of complex external and internal factors, the established system enables the interaction analysis of various substances with receptors under physiological conditions. In contrast, these disturbances are required to understand the complex machinery of cellular processes in vivo. Hence, an expression platform, on the basis of HEK293 cells, was established to study not only the interaction of integrin αIIbβ3 with cytoskeletal networks, but also the impact of mutations on integrin resulting in a disease-like phenotype. Mutations known to induce Glanzmann thrombasthenia (GT) symptoms, were introduced and led to different mechanical properties of integrin-expressing cells, especially during cell adhesion cells. Thereby, generation of biological and medically-relevant processes combined with the biophysical setup contribute to understand disease mechanisms as well as the action of therapeutic agents in diseases such as GT and ITP.
Drug-induced activation of integrin alpha IIb beta 3 leads to minor localized structural changes
(2019)
Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbβ3. Although the dramatic rearrangement of the overall structure of αIIbβ3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbβ3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism pectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbβ3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbβ3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.