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Activated Protein C Ameliorates Chronic Graft-Versus-Host Disease by PAR1-dependent Biased Cell Signaling on T-cells

Ranjeet K. Sinha, Ryan Flynn, Michael Zaiken, Katelyn Paz, Amanda L. Gavin, David Nemazee, José A. Fernández, Xiao Xu, John H. Griffin and Bruce R. Blazar

Key Points

  • * Recombinant wild type activated protein C (APC) or signaling-selective APC variants therapy reduces murine chronic GVHD pathology

  • APC’s biased, PAR1-dependent signaling due to R46 cleavage in PAR1 on donor T-cells is required for APC’s in vivo benefits in chronic GVHD

Abstract

Soluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of Activated Protein C's (APC) anticoagulant, anti-inflammatory and anti-apoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild type (WT)-APC to mice with established chronic GVHD (cGVHD), a less inflammatory, autoimmune-like disease. WT-APC normalized bronchiolitis obliterans-induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC or 5A-APC) with normal cytoprotective properties but greatly reduced anticoagulant activity provided similar results. Mechanistically, wt-APC and signaling-selective variants reduced Tfollicular helper cells, germinal center formation, immunoglobulin and collagen deposition. WT-APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T-cells from WT-PAR1 or mutated Gln41-PAR1 but not mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced non-canonical cleavage at Arg46-PAR1 known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, while anti-inflammatory and cytoprotective cell signaling by APC is essential. Since a phase II ischemic stroke clinical trial raised no safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy.

  • Submitted December 3, 2018.
  • Revision received June 24, 2019.
  • Accepted June 10, 2019.