Abstract 4927: Thrombin Activation of Proteinase-Activated Receptor 1 (PAR1) Induces Ca 2+ -Independent Vasoconstriction by Activating the Production of Reactive Oxygen Species in the Pulmonary Artery

Abstract

Thrombotic pulmonary arteriopathy is associated with ~50% of primary pulmonary artery hypertension (PAH). Thrombin and its receptor PAR 1 are thus suggested to play a critical role in the pathogenesis and pathophysiology of PAH. However, the role of thrombin in the regulation of pulmonary artery contraction still remains largely unknown. This study thus aimed to elucidate the contractile mechanism of thrombin in the isolated porcine pulmonary artery. In the presence of extracellular Ca 2+ ([Ca 2+ ] o ), thrombin induced a sustained contraction with a small sustained elevation of [Ca 2+ ] i and an increase in myosin light chain (MLC) phosphorylation. The contractile effect of thrombin was abolished by either a protease inhibitor or a PAR 1 antagonist, while it was mimicked by an agonist peptide for PAR 1 , but not PAR 4 . In the absence of [Ca 2+ ] o , thrombin induced a sustained contraction to an extent similar to that seen in the presence of [Ca 2+ ] o , but no significant increase in [Ca 2+ ] i or MLC phosphorylation was observed. A similar Ca 2+ -independent contraction was induced by H 2 O 2 . Pretreatment with N -acetyl cysteine substantially inhibited the Ca 2+ -independent contraction induced by thrombin and H 2 O 2 . The fluorescence imaging of reactive oxygen species (ROS) revealed the thrombin- induced production of ROS in the absence of [Ca 2+ ] o . When a scavenger enzyme glutathione peroxidase was suppressed, the ROS production by thrombin was observed both in the presence and absence of [Ca 2+ ] o , thus suggesting that thrombin is capable of producing ROS regardless of the presence or absence of [Ca 2+ ] o , while this effect was veiled by the scavenger activity in the presence of [Ca 2+ ] o . Rho kinase inhibitor inhibited not only the thrombin-induced contraction in the presence of [Ca 2+ ] o , but also the Ca 2+ -independent contraction induced by thrombin and H 2 O 2 and the thrombin-induced ROS production, thus suggesting multiple roles of Rho kinase. This study thus provides the first evidence that the thrombin activation of PAR 1 induces pulmonary vasoconstriction via Ca 2+ -dependent as well as Ca 2+ -independent and ROS-mediated contractile mechanisms. PAR 1 is thus suggested to be useful as a novel therapeutic target for the treatment of PAH, especially when associated with thrombus.