Activated protein C β-glycoform promotes enhanced noncanonical PAR1 proteolysis and superior resistance to ischemic injury

Abstract

Key Points• This study demonstrates a novel mechanistic role for a specific N-linked glycan in regulating PAR1 proteolysis.• We provide the first description of an APC variant with enhanced therapeutic cytoprotective activity in vivo.Activated protein C (APC) is an anticoagulant protease that initiates cell signaling via protease-activated receptor 1 (PAR1) to regulate vascular integrity and inflammatory response. In this study, a recombinant APC variant (APC N329Q ) mimicking the naturally occurring APC-b plasma glycoform was found to exhibit superior PAR1 proteolysis at a cleavage site that selectively mediates cytoprotective signaling. APC N329Q also enhanced integrin a M b 2 -dependent PAR1 proteolysis to exert significantly improved antiinflammatory activity on macrophages compared with wild-type APC. Recent therapeutic applications of recombinant APC in ischemic stroke models have used APC variants with limited anticoagulant activity to negate potential bleeding side effects. Using a mouse model of ischemic stroke and late t-PA intervention, the neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPC PS ) was compared with an identical APC variant except for the absence of glycosylation at the APC-b sequon (mAPC PS/N329Q ).Remarkably, mAPC PS/N329Q limited cerebral ischemic injury and reduced brain lesion volume significantly more effectively than mAPC PS . Collectively, this study reveals the importance of APC glycosylation in controlling the efficacy of PAR1 proteolysis by APC and demonstrates the potential of novel APC variants with superior cytoprotective signaling function as enhanced therapeutic agents for the treatment of ischemic stroke. (Blood. 2015;126(7):915-919)

A novel protein C–factor VII chimera provides new insights into the structural requirements for cytoprotective protease‐activated receptor 1 signaling

“…Recombinant PC FVII-82 was also sensitive to digestion by PNGase F (Fig. 1D) [21]. As expected, PC FVII-82 activated by thrombin showed similar amidolytic activity against a short chromogenic substrate to that of wild-type APC (data not shown).…”

“…PBMCs were treated with APC or APC FVII‐82 (both 20 n m ) for 3 h, and then stimulated with LPS (50 ng mL −1 ) for 18 h. Tumor necrosis factor (TNF)‐α secretion was measured by ELISA (R&D Systems, Minneapolis, MN, USA). Similarly, RAW264.7 macrophages were incubated with APC or APC FVII‐82 (20 n m ) for 3 h in HEPES buffer containing 3 m m CaCl 2 and 0.6 m m MgCl 2 prior to LPS stimulation (50 ng mL −1 ) for 18 h. TNF‐α secretion was measured by ELISA as previously described .…”

Targeting PAR1: Now What?

“…These signaling-selective APC variants were constructed to have limited anticoagulant activity by mutating N-linked glycan sites and/or residues in APC important for interaction with FVa or Protein S: 3K3A-APC (KKK191-193AAA), RR229/230AA, 5A-APC (KKK191-193AAA+ RR229/230AA), RD222/237CC, K193E, D36A/L38D/A39, L38D and L38D/N329Q [22,69–71]. The non-anticoagulant forms of APC stimulated PAR1-mediated cytoprotective signaling in vitro and exerted beneficial effects in murine models of sepsis and ischemic stroke [22,69–71] As cytoprotective-selective variants circumvent bleeding problems associated with APC’s anti-coagulant function, these variants are promising agents for clinical applications. 3K3A-APC is currently the only variant that has advanced to clinical trials.…”

Severe Thrombophilia in Idiopathic Fatal Pulmonary Embolism

“…In fact, it has been recently described that congenital disorders of glycosylation may influence the glycosylation of antithrombin and, therefore, its final secretion in antithrombin-deficient patients in which SERPINC1 has no mutations (de la Morena-Barrio et al, 2016). Glycosylation may have great relevance to the function as has been reported for antithrombin and protein C (Martínez-Martínez et al, 2012, Gleeson et al, 2015). As a consequence, all those alterations in the glycosylation of natural anticoagulants may result in a functional deficiency.…”