OC 28.3 - Direct binding of Lyn to GPIb beta transmembrane/cytoplasmic domain transmits platelet activation signal and regulates GPIb alpha–VWF binding

Background: The platelet glycoprotein (GP) Ib-IX, crucial for adhesion to von Willebrand factor (VWF), interacts with thrombin, influencing thrombosis and inflammation. Its ligand binding initiates platelet activation, involving Src family kinase (SFK) Lyn, associated with the GPIb-IX complex in platelet lysates. However, the specifics of Lyn's direct association, its binding site on GPIb-IX, and its essential role in Lyn activation and GPIb-IX signaling are unclear.

Aims: To determine if Lyn directly binds to GPIb-IX and locate the binding site, if confirmed.

Methods: In our study, we developed the MBP pull-down assay to identify Lyn's binding site on GPIb-IX and synthesized the peptide mPLbeta accordingly. We examined mPLbeta's impact on Lyn-GPIb-IX binding during platelet activation, its effect on SFK Y397 and Lyn Y397 phosphorylation, and its influence on GPIbalpha-induced mechanosignaling and platelet functions, including aggregation, secretion, and adhesion. Using BFP and fBFP techniques, we assessed intracellular calcium and extracellular VWF A1-GPIb alpha interactions. Additionally, we investigated mPLbeta's role in thrombus formation using the FeCl3-induced mouse carotid artery model and its impact on hemostasis with a tail bleeding assay.

Results: We show that Lyn directly interacts with the GPIb beta-chain between at the transmembrane domain and cytoplasmic domain interface. We synthesized a myristoylated peptide mPLbeta based on the binding site and demonstrated that it inhibits Lyn binding to GPIb using purified proteins and in platelets. mPLbeta inhibited GPIb-IX-dependent Lyn activation, VWF-A1 binding to GPIbalpha, intraplatelet calcium signaling induced by pulling on GPIbalpha, as well as human/mouse platelet activation and stable platelet adhesion to VWF with or without shear stress. Furthermore, mPLbeta inhibited thrombosis in vivo with a mild effect on tail bleeding.

Conclusion(s): These data demonstrate that direct binding of Lyn to GPIbbeta mediates GPIb-IX dependent platelet activation, and that targeting the Lyn-GPIbbeta interaction has the potential for treating GPIb-IX-dependent thrombosis and inflammation.