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

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.

Figure 1.

Novel C57BL/6 mice strains carrying either Arg41Gln or Arg46Gln mutations in PAR1 were generated. (A) C57BL/6 embryonic stem cells were used to generate mutations in mice using homologous recombination methods to incorporate the targeting vector pBS-FRT-PGK-NEO-FRT, as outlined in the panel and as described in supplemental Data. (B-C) To identify genotype for offspring from mating of QQ41 and QQ46 heterozygotes, PCR assays gave clear indication of genotypes, namely RR, RQ, or QQ, as shown for residues (B) 41 and (C) 46, respectively (see supplemental Data for methods). The DNA gel images were obtained from Bio-Rad GELDOC XR equipped with the image software Quantity One from Bio-Rad (Hercules, CA). (D) To record fetus development as observed at days 10 (E10) and 12 (E12) following plug formation, pictures of Wt and QQ41 embryos were taken under normal lighting. At E10, some QQ41 embryos were significantly smaller and underdeveloped compared with Wt embryos; however, no systematic analysis of embryogenesis was made.

Figure 2.

Arg41Gln and Arg46Gln PAR1 point mutations cause the predicted cellular responses to thrombin, TRAP, and APC as determined using microvascular BECs. (A) After BECs from QQ41-PAR1 mice or Wt mice were seeded in 384 well plates and the calcium dye Fluo-8 AM was added and incubated for 30 minutes, calcium release was triggered by addition of 20 nM thrombin (final concentration) (blue lines), ionomycin (purple lines), or control buffer (black lines), and calcium flux was recorded over 240 seconds. Solid lines represent results for BECs from Wt mice and dashed lines represent data for BECs from QQ41-PAR1–homozygous mice. (B) Area under curves (AUC) for calcium release (seen in panel A) were plotted for different doses of thrombin, which showed that thrombin gave the predicted dose-dependent increase in calcium ion release in Wt BECs whereas thrombin failed to initiate ion flux in BECs from QQ41-PAR1 mice. (C) Calcium flux was determined as described in “Materials and methods” following addition of different doses of a 10-mer TRAP10 and of mouse (m) or human (h) thrombin at varying doses to BECs from QQ41-PAR1 mice or Wt mice. Data points represent the normalized relative integrated total ion flux (RFU) increase that was recorded over 4 minutes compared with controls. Error bars represent standard error of the mean (SEM) for at least 3 separate experiments. Significance was analyzed using 2-way ANOVA. *P < .05. (D) Time-dependent changes in TER caused by thrombin (2.5, 5, and 10 nM) were recorded for BECs from Wt mice and QQ41-PAR1 mice. (E) The changes in TER caused by 20 μM TRAP10 for cultured BECs obtained from Wt mice and QQ-PAR1 mice were recorded. (F-G) Phosphorylation of Ser473 in Akt in BECs obtained from Wt mice and QQ41-PAR1 mice caused by murine APC (mAPC; 90 nM final in panel F) or BECs obtained from Wt mice and QQ46-PAR1 mice was determined. Total Akt antigen levels were used as loading controls to determine the ratio of phosphorylated Akt (pAkt)/Akt, which was normalized to 1.0 for no APC at zero time. Blots were scanned on LICOR and quantified error bars represent SEM for at least 3 separate experiments. Significance was analyzed using 2-way ANOVA. CI, confidence interval.

Figure 3.

APC-mediated mortality reduction in bacterial sepsis is seen for QQ41-PAR1 mice but not for QQ46-PAR1 mice. The prosurvival effect of 5A-APC in E coli–induced pneumonia sepsis was tested in Wt and in PAR1 genetically modified mice. Sepsis was induced by instillation of 10⁶ CFU E coli intratracheally. 5A-APC IV treatment was given at 15 minutes before E coli instillation and at 6 hours after infection. QQ41-PAR1 mice, QQ46-PAR1 mice, and Wt littermate controls were observed for survival following E coli infection and 5A-APC treatment over 7 days. Significance was analyzed using log rank. *P < .01.

Figure 4.

APC-mediated neuroprotection in ischemic stroke is observed in QQ41-PAR1 mice but not in QQ46-PAR1 mice. Data (mean values) for 24 hours after MCAO are shown for infarct volume (A), edema (B), foot-fault test (C), and forelimb-asymmetry test (D) for homozygous QQ41-PAR1 mice, homozygous QQ46-PAR1 mice, and the corresponding Wt controls treated with murine recombinant 3K3A-APC (0.04 mg/kg given IV 4 hours after MCAO) or vehicle. Bars indicate mean ± standard deviation (SD), n = 4-6 mice per group. Data for 24 hours after MCAO are seen for degenerating neurons determined by Fluoro-Jade C stain (E-F) and for fibrin deposition determined by anti-fibrin antibodies (E,G) for each mouse group (panel E, original magnification ×20). Treatment of mice with 3K3A-APC or vehicle is indicated by plus or minus signs under panels A-D, F, and G. Student t test and 1- or 2-way ANOVA followed by a post hoc Tukey test were used to determine statistically significant differences. The curves of functional recovery after stroke were compared using repeated-measures ANOVA. P < .05 was considered statistically significant.