Abstract 15686: Coagulation-Independent Effects of Factor Xa on Protease-Activated Receptor 2 (PAR-2) in Pulmonary Hypertension

Abstract

Factor Xa (FXa) is a key molecule in the coagulation cascade. Besides, FXa exerts direct effects on cells via protease-activated receptor 2 (PAR-2), inducing proliferation and migration. Pulmonary hypertension (PH) is a fatal disease, defined by increased pulmonary vascular resistance, causing pulmonary arterial pressure elevation and finally right heart failure. Aberrant proliferation and migration of smooth muscle cells (SMCs) are crucial for underlying vascular remodeling processes in the pulmonary vasculature. We hypothesize that FXa contributes to vascular remodeling in PH via PAR-2 in pulmonary arterial SMCs (PASMCs). In vitro proliferation was assayed via BrdU incorporation, migration via modified Boyden chamber, respectively. Human PASMCs (hPASMCs), as well as murine PASMCs (mPASMCs) from WT and PAR-2-/- mice were used. FXa (0.1-1.5U/ml) and the following inhibitors were applied: Vorapaxar (PAR-1), U0126 (ERK), and SKI-178 (sphingonsine-kinase-1). Adult male WT and PAR-2-/- mice were exposed to normoxia vs. hypoxia to develop PH. After 21 days, right ventricular systolic pressure (RVSP) was measured. Right heart hypertrophy was determined by ratio of right ventricle to left ventricle plus septum (RV/(LV+S)). FXa mediated hPASMC proliferation (2.78±0.60 fold vs. ctrl, n>4) via ERK and sphingosine-kinase-1. Whereas, vorapaxar did not affect FXa-mediated proliferation in hPASMCs (n=3). FXa-induced proliferation was significantly impaired in PAR-2-/- vs. WT PASMCs (1.41±0.11 vs. 1.79±0.05, n=6). FXa induced chemotaxis of hPASMCs (1.46±0.13 fold vs. ctrl, n=6). PAR-2-/- PASMCs displayed impaired basal migratory responses (1.45±0.46 cells) and decreased chemotaxis towards FXa vs. WT cells (1.37±0.81 vs. 9.958± 0.29 cells, n=3). PAR-2 deficiency protected from hypoxia-induced elevation of RVSP compared to WT mice (30.86±1.315 mm/Hg vs. 40.38±1.257 mm/Hg; n≥7). However, RV/(LV+S) was not decreased in PAR-2-/- vs. WT animals (0.392±0.026 vs. 0.413±0.0142; n≥7). Conclusively, PAR-2 plays an important role in experimental PH in mice. Additionally, we elucidated that F-Xa exerts direct mitogenic and chemotactic effects in PASMCs via PAR-2 activation, indicating a direct link between F-Xa and PAR-2 in pulmonary hypertension.