Advertisement

PAR1 biased signaling is required for activated protein C in vivo benefits in sepsis and stroke

Ranjeet K. Sinha, Yaoming Wang, Zhen Zhao, Xiao Xu, Laurent Burnier, Naveen Gupta, José A. Fernández, Greg Martin, Sergey Kupriyanov, Laurent O. Mosnier, Berislav V. Zlokovic and John H. Griffin

Key Points

  • R41Q and R46Q point mutations in PAR1 in mice enabled studies of APC’s in vivo mechanism of action in lethal sepsis and ischemic stroke.

  • APC-biased, PAR1-dependent signaling due to cleavage at R46 in PAR1 is required for APC’s in vivo benefits in sepsis and ischemic stroke.

Publisher's Note: There is a Blood Commentary on this article in this issue.

Abstract

Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 (F2r gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1+/R46Q mice generated expected numbers of PAR1+/+, PAR1+/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1+/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1+/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in Escherichia coli–induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice (P < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.

  • Submitted October 20, 2017.
  • Accepted December 28, 2017.
View Full Text