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Immunology
Protective Signaling by the Protein C Pathway
M. Riewald, C. Feistritzer,
J.K. Bowden-Verhoek
The
serine proteases thrombin and activated protein C (APC) are major regulators of the blood coagulation
system. Thrombin not only initiates fibrin formation and platelet activation but also activates
protein C on the surface of endothelial cells. The generated APC inhibits blood coagulation by
downregulating prothrombin activation in a negative feedback loop. Endothelial cell protein
C receptor (EPCR) can bind both protein C and APC, and activation of protein C is enhanced when the
protein is bound to the receptor. Results from animal models and clinical trials indicate that
APC has potent protective effects in systemic inflammation that are independent from its well-established
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.
We identified protease-activated receptor
1 (PAR-1), the prototypical thrombin receptor, and EPCR as part of a novel APC signaling pathway
in endothelial cells (Fig. 1).
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| Fig.
1. PAR-1 signaling by the interdependent proteases thrombin
and APC. Thrombin can have proinflammatory effects on endothelial cells, whereas APC signaling
is protective. PAR-1 activation is required for signaling by both proteases, and EPCR cosignaling
may modify responses to APC–PAR-1 signaling. Thrombin activates protein C, and APC downregulates
thrombin formation in a negative feedback loop. A change in the activity of either protease in vivo
will thus affect the activity of the other protease. |
The G-protein–coupled PAR-1 is activated by proteolytic
cleavage of a specific peptide bond in the amino-terminal exodomain, leading to the exposure of
a new amino terminus that folds back and activates the receptor. We found that APC binding to EPCR
is required for PAR-1 activation by APC, and PAR-1 activation accounted for all effects of APC on
gene expression. Large-scale gene expression profiling indicated that although PAR-1 activation
is required for endothelial cell signaling by thrombin as well as by APC, both proteases can have
distinct effects on gene expression in endothelial cells perturbed by inflammatory cytokines.
Importantly, our data indicate that signaling by EPCR–APC–PAR-1, but not by thrombin–PAR-1,
leads to downregulation of several proapoptotic genes, including p53. We are analyzing
how EPCR cosignaling may modulate distinct PAR-1–dependent effects of APC on both gene expression
and the regulation of the barrier function of an endothelial cell monolayer.
To test the hypothesis that APC–PAR-1
signaling in endothelial cells limits inflammatory responses and reduces lethality in sepsis,
we are analyzing the effect of administration of APC in mouse models of sepsis. Mutagenesis studies
of PAR-1 residues flanking the activating cleavage site led to the design of PAR-1 variants that
are efficiently activated by APC but not by thrombin and vice versa. We are currently generating
transgenic mice with Tie2 promoter–driven expression of PAR-1 variants in endothelial
cells. We expect that analysis of these mice in models of sepsis will define the in vivo role of the
EPCR–APC–PAR-1 signaling cascade in systemic inflammation.
Publications
Riewald, M., Petrovan, R.J., Donner,
A., Ruf, W. Activated protein C signals through the thrombin
receptor PAR1 in endothelial cells. J. Endotoxin Res. 9:317, 2003.
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