Involvement of reactive oxygen species in thrombin-induced pulmonary vasoconstriction

Rationale: Pulmonary vascular thrombosis and thrombotic arteriopathy are common pathological findings in pulmonary arterial hypertension. Thrombin may thus play an important role in the pathogenesis and pathophysiology of pulmonary arterial hypertension. Objectives: The present study aimed to elucidate the contractile effect of thrombin in the pulmonary artery and clarify its underlying mechanisms. Methods: The changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i), 20-kD myosin light chain (MLC20) phosphorylation, and contraction were monitored in the isolated porcine pulmonary artery. The production of reactive oxygen species (ROS) was evaluated by fluorescence imaging. Measurements and Main Results: In the presence of extracellular Ca²⁺, thrombin induced a sustained contraction accompanied by an increase in [Ca²⁺]_i and the phosphorylation of MLC20. In the absence of extracellular Ca²⁺, thrombin induced a contraction without either [Ca²⁺]_i elevation or MLC20 phosphorylation. This Ca²⁺- and MLC20 phosphorylation-independent contraction was mimicked by hydrogen peroxide and inhibited by N-acetyl cysteine. Fluorescence imaging revealed thrombinto induce the production of ROS. A Rho-kinase inhibitor, Y27632, inhibited not only the thrombin-induced Ca²⁺- and MLC20 phosphorylation-dependent contraction, but also the Ca²⁺- and MLC20 phosphorylation-independent contraction and the ROS production. These effects of thrombin were mimicked by a proteinase-activated receptor 1 (PAR₁)-activating peptide. Conclusions: This study elucidated the Ca²⁺- and MLC20 phosphorylation-independent ROS-mediated noncanonical mechanism as well as Ca²⁺- and MLC20 phosphorylation-dependent canonical mechanism that are involved in the thrombin-induced PAR₁-mediated pulmonary vasoconstriction. Rho-kinase was suggested to play multiple roles in the development of thrombin-induced pulmonary vasoconstriction.