Expression and Purification of the Glycoprotein Ibα for Structural Biology Applications

At sites of vascular injury, platelets are recruited and activated to start thrombus formation. This process is possible due to the interaction of the platelet receptor GPIbα and endothelial bond von Willebrand Factor. This interaction not only activates platelets but also generates an inflammatory response as the complex interacts with the leukocyte receptor Mac-1. However, the mechanisms behind these interactions are not well understood. The conformational changes taking place after GPIb-vWF binding and how it activates the immune system is still to be confirmed. Hence, a stable expression platform of GPIb that can be used to study its interactions with vWF and Mac-1 was established. HEK 293 GnTI-/- cells were transfected with the plasmid carrying GPIbα and selected using G418 antibiotic. Collected protein samples were purified using His Tag and Size Exclusion chromatography. Later, thermodynamic kinetics of unfolding were calculated using differential scanning fluorimetry. It was confirmed that GPIbα has a higher stability in HEPES buffers containing monovalent metals, such as NaCl and KCl. This initiative lays the foundation for investigating the intrinsic interactions of GPIbα with its ligands. Future studies of GPIbα will not only advance our understanding of platelet function and thrombo-inflammation but also offer valuable guidance for structure-dependent drug discovery under physiologically relevant conditions. By elucidating the molecular mechanisms underlying these interactions, a targeted therapeutic intervention that modulates thrombotic and inflammatory responses could be developed, which will improve clinical outcomes in conditions such as cardiovascular disease and thrombosis.