Journal
BLOOD
Volume 131, Issue 11, Pages 1163-1171Publisher
AMER SOC HEMATOLOGY
DOI: 10.1182/blood-2017-10-810895
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Funding
- National Institutes of Health, National Heart, Lung, and Blood Institute [HL104165, HL130678]
- National Institute of Neurological Disorders and Stroke [NS090904]
- National Heart, Lung, and Blood Institute [HL031950, HL052246, HL133728]
- Fondation Suisse pour les Bourses en Medecine et Biologie
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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.
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