Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar

Blood. 2015 Mar 19;125(12):1976-85. doi: 10.1182/blood-2014-09-599910. Epub 2015 Jan 13.

Abstract

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand-binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein-coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Binding Sites
  • COS Cells
  • Chlorocebus aethiops
  • Cytoplasm / metabolism
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / injuries*
  • Exocytosis
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Inflammation
  • Lactones / adverse effects*
  • Ligands
  • Platelet Aggregation Inhibitors / chemistry
  • Protein C / chemistry
  • Pyridines / adverse effects*
  • RNA, Small Interfering / metabolism
  • Receptor, PAR-1 / antagonists & inhibitors*
  • Signal Transduction
  • Thrombosis / drug therapy*
  • Thrombosis / prevention & control*

Substances

  • Lactones
  • Ligands
  • Platelet Aggregation Inhibitors
  • Protein C
  • Pyridines
  • RNA, Small Interfering
  • Receptor, PAR-1
  • vorapaxar