Studying platelet and monocyte function in atherosclerosis by employing quantitative proteomics

Atherosclerosis is the major cause of cardiovascular diseases. Both monocytes and platelets are key players in the development and progression of atherosclerosis. Monocyte adhesion, transmigration, and differentiation contribute to atherosclerotic plaque formation. Activated platelets release their granule contents, platelet releasate, to activate and modulate monocytes. The platelet releasate consists of a large number of bioactive proteins, including multiple cytokines and chemokines. The binding of chemokines to specific G protein-coupled receptors in the plasma membrane of monocytes can initiate the intracellular signaling cascade that results in monocyte migration. Multiple small GTPases are critical elements in such intracellular signaling pathways. To determine the effect of platelet releasate on small GTPases in monocytes, the activity level changes of small GTPases were quantified by the quantitative multiplexed small GTPase activity assay. Upon lysophosphatidic acid (LPA)-induced platelet releasate treatment, the activation of both Rap1 and Rap2 GTPases in monocytes was found to be increased through chemokine CXCL12 binding. Like LPA, oxidized phosphatidylcholines (oxPCs) are also oxLDL derivatives. They can be released from plaques upon rupture. The effect of oxPCs on platelets is controversial, but involves the cAMP/cGMP signaling pathway. To define the response of the cAMP/cGMP interactome in platelets upon oxPC treatment, a targeted quantitative chemical proteomics approach was developed and applied to purify cAMP/cGMP binding proteins and quantify their amount change in platelets upon KDdiA-PC and POV-PC stimulation. The amount of PKA-RIα and -RIIα was decreased in POV-PC-stimulated platelets, which coincides with an increase in the intracellular cAMP level. However, sGC inhibitor treatment further decreased the amount of PKA-RIβ and -RIIβ, and PKG1 in POV-PC stimulated platelets, which implies the reduction of intracellular cGMP upon POV-PC stimulation and the cross-talk between cAMP and cGMP signaling. The interaction between phosphodiesterases and cAMP/cGMP demonstrates the compartmentalization of cyclic nucleotide signaling. These findings may help recognize therapeutic targets for prevention of inappropriate monocyte activation and contribute to the application of platelet drugs in combination to avoid undesirable side effects like bleeding.Science, Faculty ofChemistry, Department ofGraduat