Development and application of proteomic tools to study platelet function in the context of cardiovascular disease

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Recent evidence suggests that activated platelets play an important role in promoting the recruitment of leukocytes, particularly monocytes, to damaged endothelium, which leads to formation of atherosclerotic plaques. Lysophosphatidic acid (LPA) accumulates in atherosclerotic plaques, activates additional platelets and amplifies immune response. Therefore, understanding the molecular mechanism of how platelets are activated and the effect of platelet-monocyte aggregate (PMA) formation is key to the design of intervention strategies for CVDs. To achieve this, two novel proteomics tools, i.e. in silico protein interaction analysis and quantitative multiplexed small GTPase activity assay, were developed and applied to study platelet functions. Using the former approach, a core network involved in platelet aggregation was identified that consists of integrin αIIb, integrin β3, talin1, fibrinogen α and β chains, Rap1b and other cytoskeletal proteins. Using the latter method, time-resolved activation profiles of ten small GTPases, i.e. Ras, Rho, Rap and Rac isoforms, in platelets in response to thrombin, ADP and LPA were generated. The addition of PI3K inhibitors only reduced the activation levels of Rap1A and Rap1B without affecting other small GTPase isoforms in LPA-induced platelet activation. Moreover, the small GTPase Rac and calcium, but not PI3 kinase or the small GTPase Rho, were found to be key regulators for LPA-induced platelet secretion. Furthermore, PMA formation was significantly increased in THP-1 monocytic cells incubated with thrombin- or LPA-activated platelets. To investigate the signaling changes in this process systematically, a quantitative phosphoproteomics strategy was adapted and optimized for a two-cell system, which revealed several key biological processes in monocytes, including leukocyte activation, small GTPase activation and cytoskeleton organization. In summary, the research in this thesis has furthered our understanding of platelet function in the context of cardiovascular disease, which might serve as a basis for designing more targeted approaches for antiplatelet therapies. In addition, the proteomics tools we have developed and validated now enable the exploration of new avenues of research, as demonstrated by their successful application to study biological systems.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat