Shear and Integrin Outside-In Signaling Activate NADPH-Oxidase 2 to Promote Platelet Activation

Zheng Xu, Ying Liang, M. Keegan Delaney, Yaping Zhang, Kyungho Kim, Jing Li, Yanyan Bai, Jaehyung Cho, Masuko Ushio-Fukai, Ni Cheng, Xiaoping Du

Research output: Contribution to journalArticlepeer-review

Abstract

Objective: Despite the importance of reactive oxygen species (ROS) and NOX (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) 2 in platelet activation and in vivo thrombosis, it is unclear how ROS and NOX2 play a role in platelet activation and why NOX2 deficiencies in humans and mice do not affect hemostasis. Outside-in signaling of integrin αIIbβ3mediates platelet response to shear stress, secondary platelet activation, and thrombus expansion and is critical to thrombosis but dispensable for hemostasis. We studied the mechanisms of platelet ROS generation, ROS-mediated platelet response, and the role of ROS in integrin αIIbβ3outside-in signaling. Approach and Results: ROS generation in activated platelets was low and slow without shear but was robust under shear. Shear-enhanced ROS generation and activation of p47phox, an important regulatory subunit of NOX2, were diminished by the integrin antagonist integrilin or β3knockout, and by Gα13knockout or blocking the Gα133interaction. Resting platelets spreading on integrin ligand fibrinogen also Gα13-dependently stimulated ROS generation and p47phox activation. Hence, Gα13-mediated outside-in signaling induces NOX2 activation and ROS generation which is greatly enhanced by shear. Outside-in NOX2 activation requires Src, phosphoinositide 3-kinase and Akt downstream of Gα13. Importantly, NOX2-knockout platelets showed defective ROS generation, reduced platelet spreading without shear, and reduced platelet adhesion and thrombus volume on collagen and VWF (von Willibrand factor) under shear, whereas ROS inhibition diminished activation of tyrosine kinase Syk. Conclusions: Outside-in signaling activates the mainly NOX2-mediated ROS generation, which mediates Syk-dependent secondary platelet activation, adhesion, and thrombosis with minimal effect on hemostasis.

Original languageEnglish (US)
Pages (from-to)1638-1653
Number of pages16
JournalArteriosclerosis, thrombosis, and vascular biology
DOIs
StateAccepted/In press - 2021

Keywords

  • glycoprotein
  • integrin
  • platelet activation
  • reactive oxygen species
  • thrombosis

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

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