Doctoral researcher: Sanjai Karanth
Principle investigator: Mihaela Delcea
Co-supervisor: U. Bornscheuer
The role of oxidizing agents in alphaIIbbeta3 integrin activation
Integrins are an important family of transmembrane adhesion receptors that mediate cell adhesion to the extracellular matrix proteins, or cell-cell interaction. AlphaIIbbeta3(αIIbβ3) is the most abundant integrin heterodimer on blood platelets with an average of 80,000 copies per cell .
Crystal structures revealed that low-affinity state integrins are in a bent conformation with their ligand binding sites placing in proximity to the membrane surface . Intermediate extended conformations with a closed head correspond to activated state integrin, where the conformational changes in the extracellular domain lead to increased ligand affinity. Binding of ligand induces further conformational changes, resulting in a high-affinity extended open conformation that fully activates integrin signaling downstream . αIIbβ3 plays a crucial role in haemostasis and is involved in the autoimmune disease Immune Thrombocytopenia (ITP) characterized by low platelet count and bleeding risks. Although it has been suggested that oxidants such as hydrogen peroxide may play a role in ITP , it is unknown whether such oxidants activate αIIbβ3.
We aim to study the influence of oxidants derived from platelets on the activation of αIIbβ3. For this purpose, αIIbβ3 will be reconstituted in a membrane environment (lipid bilayers) and will let to interact with exogenous oxidants and their products (e.g. hydrogen peroxide, H2O2; superoxides, O2-; nitric oxide, .NO).
Protein modification (activation) as a result of oxidative damage will be investigated using a combination of biophysical techniques. Liposome preparation and αIIbβ3 incorporation will be carried out and characterized by quartz crystal microbalance (QCM) and atomic force microscopy (AFM).
Förster resonance energy transfer (FRET) will be used to prove whether or not αIIbβ3 is activated. In addition, antibodies which recognize the activated αIIbβ3 will be used in an enzyme immunosorbent assay to validate protein activation by oxidants. The interaction of oxidants and their products with αIIbβ3 will be also investigated under various pH, ions and temperature conditions (Fig.1).
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