Manifest destiny at the Scripps Research Institute

Abstract 534 Activated protein C (APC) exerts direct cytoprotective activities on cells that are required for APC's ability to reduce mortality in murine sepsis models and that likely have potential contributions to mortality reduction in patients with severe sepsis. Although multiple receptors have been implicated to promote APC direct cytoprotective activities on various cell types, the endothelial protein C receptor (EPCR) and PAR1 play pivotal roles in our current understanding of the molecular mechanisms for APC's direct effects on cells. According to the current generally accepted model, binding of APC to EPCR on the endothelial cell membrane facilitates activation of PAR1 and induction of signaling that ultimately results in cytoprotective effects. However, this model raises the dilemma of how thrombin and APC can often have seemingly opposing effects when activating PAR1. To gain insights into mechanisms for the contrasting effects of APC vs. thrombin signaling, PAR1 activation by APC was studied in greater detail. We hypothesized that APC might cleave PAR1 not only at Arg41 but also at Arg46 with distinctly different consequences than caused by Arg41 cleavage and that this alternative cleavage could distinguish APC's from thrombin's effects. Previously we found that, in the presence of EDTA, APC cleaved a synthetic PAR1 N-terminal peptide (TR33-66) at Arg41. Now we have found that, in buffers containing CaCl2, APC cleaved the PAR1 TR33-66 peptide at Arg41 and also at an additional site distal from Arg41. Proteolysis of the TR33-66 peptide with APC resulted in fragments corresponding to TR33-41 and TR42-66 similar to thrombin. But in contrast to thrombin, a third fragment was generated by APC and the TR42-66 fragment disappeared over time with the concomitant accumulation of a novel peptide. Furthermore, incubation of thrombin-cleaved TR33-66 with APC resulted in additional proteolysis of TR42-66 and generation of the same novel fragment, indicating the possibility of a second APC cleavage site in PAR1 that was distinct and distal from Arg41. Isolation of the novel proteolytic fragments and their MALDI-TOF analysis identified Arg46 as the second APC cleavage site in the TR33-66 peptide. When cells containing wild-type EPCR were transfected with SEAP-PAR1 wild type and mutant constructs, both thrombin and APC cleaved wt-PAR1. As anticipated, efficient cleavage by thrombin was observed for R46Q-PAR1 but not R41Q-PAR1 or R41Q/R46Q-PAR1. In contrast, APC readily cleaved both R41Q-PAR1 and R46Q-PAR1 whereas cleavage of R41Q/R46Q-PAR1 by APC was negligible. APC mediated cleavage of R41Q-PAR1 and R46Q-PAR1 required the presence of functional EPCR and was not supported by the APC-binding-defective E86A-EPCR mutant. These results indicate that on cells Arg46 in PAR1 can serve as a second cleavage site for APC. Since the new PAR1 N-terminus after proteolysis acts as a tethered ligand for receptor activation, cleavage at Arg41 vs. Arg46 could potentially create structurally distinct agonists, which might help explain the divergent patterns for PAR1-mediated cytoprotective APC signaling vs. proinflammatory IIa signaling. In studies to see if the APC-induced new PAR1 N-terminus starting at Asn47 could promote signaling, we found that a synthetic peptide with the PAR1 47–66 N-terminal sequence (NPND-peptide) increased Akt phosphorylation at Ser473 in endothelial cells over 30 min whereas neither a control scrambled sequence (47-66)-peptide nor a TRAP peptide had a similar remarkable effect on Akt phosphorylation. Moreover, the NPND-peptide, but neither the scrambled sequence-related peptide nor a TRAP peptide, inhibited staurosporine-induced endothelial cell apoptosis. Thus, it appears that the new N-terminus generated by APC's cleavage at Arg46 in PAR1 generates a novel tethered ligand that can induce cytoprotective APC-like but not thrombin-like signaling characteristics. In summary, APC is capable of a unique, functionally significant cleavage of PAR1 at Arg46 that can initiate distinctive signaling compared to canonical cleavage of PAR1 at Arg41. This implies activated PAR1 is a GPCR with multiple active conformations capable of multiple, agonist selective activity profiles which may help explain the divergent patterns for cytoprotective APC signaling vs. proinflammatory thrombin signaling. Disclosures: Mosnier: Scripps Research institute: Employment, Patents & Royalties. Griffin:ZZBiotech LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Scripps Research Institute: Employment, Patents & Royalties.

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Novel R41Q-PAR1-Modified Mice Enable Proof-of-Concept Studies for in Vivo Mechanisms of Action for Thrombin (IIa) and Activated Protein C (APC)

Blood ◽

10.1182/blood.v124.21.99.99 ◽

2014 ◽

Vol 124 (21) ◽

pp. 99-99

Author(s):

Ranjeet Kumar Sinha ◽

Laurent Burnier ◽

Naveen Gupta ◽

Xiao Xu ◽

Sergey Kupriyanov ◽

...

Keyword(s):

Protein C ◽

Activated Protein C ◽

Mutant Mice ◽

Endothelial Barrier ◽

Scripps Research Institute ◽

Wild Type ◽

E Coli ◽

Homozygous Mutant ◽

Research Institute

Abstract Introduction: Thrombin (IIa) and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses that affect many cell types in the body. IIa employs the GPCR, protease activated receptor 1 (PAR1), to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC requires PAR1 for its opposing actions to stabilize endothelial barriers and to provide anti-inflammatory and anti-apoptotic activities. Studies of murine in vivo injury models using PAR1 knockout mice show that APC requires PAR1 to reduce sepsis-induced mortality and to provide robust neuroprotection following ischemic stroke. Extensive in vitro studies support the hypothesis that IIa’s cleavage at R41 in PAR1 initiates its signaling. Although APC was long thought, paradoxically, to act also via cleavage at R41, we recently proposed that APC’s cleavage at R46 initiates its endothelial barrier-protective and cytoprotective signaling via biased signaling. Since PAR1 knock-out mice cannot provide mechanistic data for testing these hypotheses in vivo, mice carrying the point mutation of R41 to Q in PAR1 were generated to enable mechanistic studies to test whether or not IIa and APC require Arg41 for their PAR1-dependent effects. Methods: Using C57BL/6-derived embryonic stem cells and standard gene targeting methods, we prepared C57BL/6 mice carrying the PAR1 mutation of R41 to Q. IIa-induced and APC-induced signaling, detected as phosphorylation of ERK1/2 or Akt in Brain Microvascular Endothelial Cells (BECs), was quantified using immunoblotting. BECs were obtained from homozygous 41QQ-PAR1 mice and wild type 41RR-littermates. Endothelial barrier disruption of cultured BECs was assayed using Trans-Endothelial Resistance (TER) assays (iCelligence, Acea, San Diego). Mortality of wild type and 41QQ-PAR1 mutant mice that was caused by live E. coli-induced pneumonia and to endotoxin was determined using standard methods. The ability of a cytoprotective-selective murine APC mutant (5A-APC) to reduce mortality of E.coli-challenged wild type and homozygous mutant mice was determined. Results: Upon breeding of R41Q-PAR1 heterozygous mice, the progeny did not fit a Mendelian pattern and yielded only 14% rather than 25% homozygous 41QQ mice. This reduced yield of homozygous mutant mice was similar to the previously reported low yield of homozygous PAR1 knockout mice. Homozygous 41QQ-PAR1 mice showed normal protein expression in BECs for PAR1 and endothelial cell protein C receptor (EPCR) antigens. When BECs from homozygous mutant mice were compared to those from wild type littermates, the IIa-induced vascular disruption in TER assays was greatly reduced by the mutation. Intracellular Ca2+ release, a hallmark of IIa-induced signaling, was greatly impaired (>90%) in BECs from homozygous mutant mice compared to wild type controls. IIa-induced phosphorylation of ERK1/2 in BECs was also significantly reduced by the mutation whereas APC-induced phosphorylation of Akt was not significantly affected. In murine sepsis-induced mortality studies, homozygosity for the R41Q-PAR1 mutation conveyed considerable resistance to death induced by either E. coli pneumonia or endotoxin in female mice but not in male mice. Tests to determine whether 5A-APC rescued male mice from sepsis-induced lethality showed that homozygous 41QQ-PAR1 mice were entirely responsive to 5A-APC therapy because 5A-APC treatment reduced mortality from 50 % to 0 % (see Figure). Wild type control mice also showed a beneficial response with reduced mortality in response to 5A-APC therapy, as previously described. Conclusions: These studies show that mutation of Arg41 to Gln in murine PAR1 diminishes or eliminates signaling induced by IIa but not by APC. Moreover, the ability of cytoprotective-selective 5A-APC to reduce bacteria-induced septic mortality in 41QQ-PAR1 mutant mice provides strong in vivo proof-of-concept data for PAR1 activation caused by non-canonical cleavage by APC. In summary, the 41QQ-PAR1 mutant mouse provides a unique and powerful tool to define in vivo requirements for cleavage sites that enable PAR1 signaling activities induced by IIa, APC or other proteases. Figure 1 Figure 1. Disclosures Mosnier: The Scripps Research Institute : The Scripps Research Institute Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties.

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