Integrin αIIbβ3 - a journey from protein to cell

I n a u g u r a l d i s s e r t a t i o n

zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.)

der Mathematisch-Naturwissenschaftlichen Fakultät

der

Universität Greifswald

vorgelegt von Una Janke

Greifswald, Dezember 2020

Dekan: Prof. Dr. Gerald Kerth 1. Gutachter: Prof. Dr. Mihaela Delcea 2. Gutachter: Prof. Dr. Anisur Rahman

Tag der Promotion: 23.03.2021

II

“The blood is life…” Bram Stoker, Roman Dracula (1897)

III

Table of Contents

Abstract ...... I

Zusammenfassung ...... II

Contribution of Others ...... III

List of Abbreviations ...... IV

1. Introduction ...... 1

1.1. Platelets and their role in health and disease ...... 1 1.2. Platelet activation ...... 2 1.3. Platelet mechanics and cytoskeleton ...... 3 1.4. Platelet membranes and lipids ...... 5 1.5. The platelet αIIbβ3 ...... 7 1.5.1. ...... 7 1.5.2. Structure of αIIbβ3 ...... 8 1.5.3. Activation and function of αIIbβ3 ...... 10 1.5.4. The role of divalent cations in integrin activation ...... 12 1.6. Biomimetic membrane systems ...... 13 1.6.1. Model membranes ...... 13 1.6.2. Liposomes ...... 14 1.7. Nanoparticles and their applications ...... 16 1.8. Biophysical tools to study proteins and their interactions with lipids, drugs and nanoparticles ...... 16 1.9. Aim of this thesis ...... 18

2. Materials ...... 19

2.1. General equipment ...... 19 2.2. Chemicals, kits and consumables ...... 21 2.3. Proteins, enzymes, nanoparticles and antibodies ...... 23 2.4. Purification procedures ...... 24 2.5. Liposome preparation ...... 24 2.6. Cell and bacterial culture ...... 25 2.7. Software ...... 26 3. Experimental procedures ...... 28

3.1. Enzyme-linked immunosorbent assay (ELISA) ...... 28 3.2. Integrin purification ...... 28

I

3.3. Integrin reconstitution into liposomes ...... 29 3.3.1. Reconstitution protocol with Triton X-100 ...... 29 3.3.2. Reconstitution protocol with CHAPS ...... 30 3.4. Characterization of liposomes ...... 30 3.4.1. Phosphate assay ...... 30 3.4.2. Thin-layer chromatography (TLC) ...... 31 3.4.3. Transmission electron microscopy (TEM) – Negative staining ...... 31 3.4.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ...... 32 3.4.5. Determination of protein concentration ...... 32 3.4.6. Dynamic light scattering (DLS) ...... 32 3.5. Activation of integrin ...... 34 3.5.1. Flow cytometry of liposomes ...... 34 3.5.2. Quartz crystal microbalance with dissipation (QCM-D) monitoring...... 34 3.5.3. Nanoparticle synthesis and modification ...... 36 3.5.4. Activation assay ...... 36 3.5.5. Molecular dynamic simulations (MDS) ...... 36 3.5.6. Circular dichroism (CD) spectroscopy ...... 37 3.6. HEK293 cells as integrin expression platform ...... 39 3.6.1. Cell culture and transfection ...... 39 3.6.2. Mutagenesis and isolation of plasmid DNA ...... 40 3.6.3. Confocal microscopy ...... 43 3.6.4. Flow cytometry and fluorescence-activated cell sorting (FACS) ...... 43 3.6.5. Atomic force microscopy (AFM) ...... 45 3.6.6. Real time-deformability cytometry (RT-DC) ...... 49 3.6.7. Statistical analysis ...... 51 4. Results ...... 52

4.1. Integrin αIIbβ3 in biomimetic systems ...... 52 4.1.1. Integrin characterization ...... 52 4.1.2. Bilayer formation of liposomes with various lipidic systems ...... 53 4.1.3. Reconstitution of integrin into liposomes ...... 55 4.1.4. Proteoliposome-derived bilayer formation ...... 56 4.1.5. Conformational changes of integrin upon treatment with manganese ...... 59 4.1.6. Secondary structure changes upon manganese-induced integrin activation ... 62 4.1.7. Platelet integrin activation induced by clinical drugs ...... 63 4.1.8. Interaction of integrin αIIbβ3 with fibrinogen bioconjugates ...... 66 4.1.9. Influence of different lipidic systems on platelet integrin activation ...... 68 4.2. HEK293 cells as an expression platform and model system to study the impact of integrin on cell elasticity ...... 75 II

4.2.1. Expression of integrin αIIbβ3 and co-localization ...... 76 4.2.2. Integrin activation and cytoskeletal rearrangements ...... 77 4.2.3. Cell elasticity and platelet integrin activation ...... 83 5. Discussion ...... 85

5.1. Liposomes as biomimetic system ...... 85 5.2. Integrin activation in liposomes ...... 86 5.3. Influence of clinically relevant drugs on integrin activation ...... 88 5.4. Interaction of integrin αIIbβ3 with fibrinogen-nanoparticle bioconjugates ...... 89 5.5. Influence of the lipidic system on integrin dynamics ...... 91 5.6. Impact of integrin on cell elasticity and cytoskeleton ...... 95 5.7. Summary and perspectives ...... 99 6. List of figures ...... 100

7. List of tables ...... 101

8. References ...... 102

9. Appendix ...... 117

Acknowledgements ...... 128

III

Abstract

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 cells. Thereby, g