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Endothelial dysfunction is associated with multiple vascular diseases and lacks effective treatment options. Activated Protein C (aPC) is a promising biotherapeutic that signals via protease-activated receptor-1 (PAR1) to promote diverse cytoprotective responses, including endothelial barrier stabilization, anti-inflammatory, and anti-apoptotic activities, which require specific co-receptors. We show that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl-2) scaffolds rather than heterotrimeric G proteins. However, the mechanisms by which aPC/PAR1 elicits diverse cytoprotective responses are poorly defined. Here we define a novel β-arrestin-2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. aPC stimulates the phosphorylation, translocation, and activation of SphK1 and is dependent on β-arrestin-2 and not Dvl-2. Moreover, aPC/PAR1 markedly increases S1PR1-caveolin-1 co-association, although both receptors co-existed in caveolin-1 enriched microdomains before aPC stimulation. These studies reveal that aPC/PAR1 cytoprotective responses are mediated by discrete β-arr2-driven signaling pathways modulated by co-receptors localized in caveolae.

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