Platelet procoagulant phenotype is modulated by a p38-MK2 axis that regulates RTN4/Nogo proximal to the endoplasmic reticulum: Utility of pathway analysis

Upon encountering physiological cues associated with damaged or inflamed endothelium, blood platelets set forth intracellular responses to ultimately support hemostatic plug formation and vascular repair. To gain insights into the molecular events underlying platelet function, we used a combination of interactome, pathway analysis, and other systems biology tools to analyze associations among proteins functionally modified by reversible phosphorylation upon platelet activation. While an interaction analysis mapped out a relative organization of intracellular mediators in platelet signaling, pathway analysis revealed directional signaling relations around protein kinase C (PKC) isoforms and mitogen-activated protein kinases (MAPKs) associated with platelet cytoskeletal dynamics, inflammatory responses, and hemostatic function. Pathway and causality analysis further suggested that platelets activate a specific p38-MK2 axis to phosphorylate RTN4 (reticulon-4, also known as Nogo), a Bcl-xl sequestration protein and critical regulator of endoplasmic reticulum (ER) physiology. In vitro, we find that platelets drive a p38-MK2- RTN4-Bcl-xl pathway associated with the regulation of the ER and platelet phosphatidylserine exposure. Together, our results support the use of pathway tools in the analysis of omics data sets as a means to help generate novel, mechanistic, and testable hypotheses for platelet studies while uncovering RTN4 as a putative regulator of platelet cell physiological responses.