Response by Liu and Hu to Letter Regarding Article, “Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase”

Abstract

In Response:We welcome the letter commenting on our recent study, which raised a question on whether the defective reactive oxygen species (ROS) generation and the impaired platelet activation associated with VPS34 (vacuolar protein sorting 34) deficiency are bona fide function of NADPH oxidase (NOX), as suggested by our study.1 Our conclusion that platelet VPS34 functions through NOX (likely the isoform NOX2) and the generation of ROS thereof is based on the observations as follows: (1) VPS34 deficiency impairs NOX2 assembly; (2) VPS34 deficiency reduces the production of ROS; (3) NOX inhibition by DPI (diphenyleneiodonium) abolishes the aggregation difference between VPS34 −/− and wild-type platelets, and supplementation of H 2 O 2 rescues the aggregation defect of VPS34 −/− platelets. Similar observation is obtained in human platelets by using VPS34 inhibitor 3-ma. These data strongly suggest that NOX2 is the main target of VPS34 in platelets although we do agree that including mice deficient in NOX2 and other NOX subunits would further clarify this point.We understand that the question mainly stems from the controversy between the previous reports on the importance of NOX for platelet activation. Hence, although the recent study using NOX1 and NOX2 deficient mice has proven that these 2 isoforms are selectively important for ROS generation and platelet activation, 2 there are previous reports showing that (1) murine platelets lacking NOX subunits, such as NOX2 or p47Phox, display normal ROS generation and aggregation responses to thrombin and collagen 3,4 ; and (2) human platelets deficient of NOX2 (from chronic granulomatous disease) also exhibit normal aggregation responses. 5,6 However, it is notable that the inhibitory effects of NOX1 and NOX2 knockout on ROS generation and platelet aggregation diminish at high concentrations of agonists.2 Consistent with the findings that VPS34 functions through NOX, VPS34 deficiency also causes an obvious inhibition of ROS production and platelet aggregation with low dose agonists stimulation (thrombin, 0.025 U/mL or collagen, 0.5 μg/mL), but the inhibition diminishes on the raise of the agonist dosage (thrombin, 0.05 U/mL or collagen, 1 μg/mL). Yet high concentrations of agonists (collagen, 30 μg/mL 3 ; collagen, 4 μg/mL 5 ; collagen, 2 μg/mL 6 ; and CRP [collagen-related peptide], 1 μg/mL 4 ) is the condition tested by the opposing studies (including the chronic granulomatous disease patient study 5,6 ) to evidence the insignificant role of NOX in platelet activation. Thus, comparison of the data obtained by different previous reports does not rule out the importance of NOX in platelet activation but rather suggests that the reliance of platelet activation on the VPS34-NOX axis is stimulation intensity-dependent.Nevertheless, both our study 1 and the study conducted by Delaney et al 2 demonstrate that low dose agonists (thrombin, 0.018 U/mL and 0.025 U/mL; collagen, 0.5 μg/mL; or CRP, 0.5 μg/mL) still activate significant ROS generation in platelets deficiency of NOX...