Scientific Report 2005

Immunology

Protective Protease-Activated Receptor 1 Signaling by the Protein C Pathway

M. Riewald, C. Feistritzer, R.A. Schuepbach, R. Lenta

Thrombin and activated protein C (APC) are major regulators of the blood coagulation system. Thrombin not only is the key procoagulant enzyme but also activates the anticoagulant protein C pathway on the surface of endothelial cells. The generated APC inhibits blood coagulation by downregulating prothrombin activation in a negative feedback loop. Results from animal models and clinical trials indicate that APC has potent protective effects in systemic inflammation that are independent of its anticoagulant function, and recently recombinant APC was approved to treat patients with severe sepsis. The molecular basis for the anti-inflammatory effects of APC is incompletely understood.

Previously, we showed that APC signaling in endothelial cells requires binding to endothelial protein C receptor and activation of protease-activated receptor 1 (PAR-1), the thrombin receptor. Thrombin–PAR-1 signaling has well established proinflammatory effects, including disruption of endothelial barrier function, raising the question of how the same receptor can also mediate protective effects of APC. Incubation of an endothelial monolayer with APC and low concentrations of thrombin potently enhanced barrier integrity through PAR-1–dependent transactivation of the barrier protective sphingosine 1-phosphate (S1P) signaling pathway (Fig. 1).

Fig. 1. PAR-1 can mediate opposite effects on endothelial barrier integrity. Inflammatory disorders such as sepsis are associated with increased permeability of the endothelial cell monolayer at the blood-tissue interface. Proinflammatory signaling by thrombin through PAR-1 can disrupt endothelial barrier integrity. APC enhanced endothelial barrier integrity dependent on binding to endothelial protein C receptor (EPCR) and activation of PAR-1, cellular sphingosine kinase-1 (SK1), and S1P receptor-1 (S1P1). Thus, barrier protection by APC proceeds via cross talk between the barrier-disruptive PAR-1 and barrier-protective S1P pathways. Incubation of cells with low concentrations (~40 pM) of thrombin had an equally potent barrier-enhancing effect dependent on the S1P pathway.

These results revealed an unexpected role for cross-communication between the prototypical barrier protective S1P and barrier-disruptive PAR-1 pathways and suggest that S1P signaling may mediate protective effects of APC in sepsis. In addition, large-scale gene expression profiling indicated that thrombin and APC can have distinct PAR-1–dependent effects in inflammatory cytokine-perturbed endothelial cells. APC–PAR-1, but not thrombin–PAR-1, downregulated transcript levels of several proapoptotic proteins, including p53 and thrombospondin-1.

Taken together, these results indicate that the same receptor, PAR-1, can mediate different biological effects depending on the rate of receptor activation, and they suggest that APC elicits powerful protective responses precisely because it is a relatively poor PAR-1 activator compared with thrombin. We are elucidating how differences in the level of receptor activation translate into activation of different cellular signaling pathways. To dissect the contributions of PAR-1 activation by APC and thrombin, we designed PAR-1 variants that are efficiently activated by APC but not by thrombin. Transgenic mice expressing these variants in endothelial cells have been generated and will be analyzed in sepsis models to define the in vivo roles of PAR-1 signaling in systemic inflammation.

Publications

Feistritzer, C., Mosheimer, B.A., Kaneider, N.C., Riewald, M., Patsch, J.R., Wiedermann, C.J. Thrombin affects eosinophil migration via protease-activated receptor-1. Int. Arch. Allergy Immunol. 135:12, 2004.

Feistritzer, C., Riewald, M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 105:3178, 2005.

Riewald, M., Ruf, W. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J. Biol. Chem. 280:19808, 2005.