Andrew Mack

Advisor: Xiaoping Du

Title: Focal Adhesion Kinases are Activated by b3 Integrins and ROS to Mediate Outside-in Signaling in Platelets

Ligand binding to integrin a_IIbb₃ mediates stable platelet adhesion and induces integrin outside-in signaling, leading to platelet spreading, granule secretion, expansion of the platelet thrombi and pathological thrombosis. These signaling events are driven by kinases to activate other platelet signaling pathways and receptors to generate maximal platelet activity. Focal adhesion kinases including Focal Adhesion Kinase (FAK) and its closely related homolog Protein Tyrosine Kinase 2-beta (PYK2) are recognized in many cell types to mediate integrin signaling following ligand binding; however, it is not clear how focal adhesion protein phosphorylation and activation is regulated and the exact roles these proteins serve during outside-in signaling in platelets. Here we show that reactive oxygen species (ROS) play a key role in integrin-dependent stimulation of FAK phosphorylation and activation, and that ROS-stimulated FAK activation is downstream of the Ga13-integrin interaction and the Src-NOX2 signaling pathway. Furthermore, ROS-dependent FAK activation is important for the integrin outside-in signaling-mediated activation of Syk and immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway. We confirmed FAK knockout in platelets (FAK^fl/fl PF4 Cre⁺) imparts defective spreading and adhesion to fibrinogen, resulting in extended tail bleeding times, yet FAK knockout has minimal impacts on other platelet functions. To this end, we tested the compensatory role of PYK2 and observed PYK2 hyperphosphorylation in stimulated FAK^fl/fl PF4 Cre⁺ platelets. Additionally, the integrin-dependent activation mechanisms of PYK2 are identical to those as FAK, indicating the ability of PYK2 to compensate for lack of FAK in outside-in signaling. Furthermore, PYK2 inhibition in FAK knockout platelets exacerbates the platelet spreading, adhesion deficiencies, and induced platelet aggregation defects unobserved in FAK knockout platelets alone. Future studies will investigate in vivo impacts of dual focal adhesion kinases inhibition as well as dual knockout in FAK^fl/fl PF4 Cre⁺ PYK2^-/- mice studies.