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

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 α IIb β 3 mediates 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 α IIb β 3 outside-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 β 3 knockout, and by Gα 13 knockout or blocking the Gα 13 -β 3 interaction. Resting platelets spreading on integrin ligand fibrinogen also Gα 13 -dependently stimulated ROS generation and p47phox activation. Hence, Gα 13 -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 Gα 13 . 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.

Aβ1–40-Induced Platelet Adhesion Is Ameliorated by Rosmarinic Acid through Inhibition of NADPH Oxidase/PKC-δ/Integrin αIIbβ3 Signaling

“…Overall, the underlying mechanisms for the inhibitory effect of RA on Aβ 1–40 -induced platelet adhesion may involve inhibition of NADPH oxidase/ROS/PKC-δ/integrin α IIb β 3 signaling. Although reports have previously indicated a relationship between NADPH oxidase and integrin α IIb β 3 activity in platelets [ 7 , 41 ], this However, the present study is the first to reveal a link between NADPH oxidase/PKC-δ and Aβ 1–40 -induced integrin α IIb β 3 in the process of platelet adhesion. Our study further showed that RA inhibits all these signaling pathways, consequently inhibiting platelet adhesion.…”

“…Platelets are key cells that play a critical role in vascular pathology via thrombogenic activity, as well as by adhering to damaged vessels [ 1 , 7 , 8 ]. Recent studies have indicated a possible role of platelets in the pathology of vascular dementia and AD, given that platelets contain amyloid precursor proteins (APP) and α-, β-, and γ-secretases, which contribute to the production of circulating Aβ [ 9 , 10 ].…”

Design of artificial vascular devices: Hemodynamic evaluation of shear-induced thrombogenicity

“…Under low shear rate perfusion in normal physiology (such as venous flow), platelets interact with adsorbed fibrinogen and collagen via their glycoprotein VI (GPVI) receptors. The integrin α IIb β 3 (also known as GPIIb/IIIa) located on platelet membranes enhances the arrest of free platelets onto immobilised fibrinogen (Savage et al, 1998;Maxwell et al, 2016;Xu et al, 2021) (Figure 5). The strong integrin α IIb β 3 -fibrinogen interaction guarantees a firm and immediate adhesion of the platelet on contact with the surface (Savage et al, 1996).…”

Self-Sustained Regulation or Self-Perpetuating Dysregulation: ROS-dependent HIF-YAP-Notch Signaling as a Double-Edged Sword on Stem Cell Physiology and Tumorigenesis

“…Note that GSK3β is also involved in the HIF-α subunit regulation ( Flugel et al, 2007 ) (Module Box I and Figures 2B,C ) and the axin-dependent YAP degradation and compartmentalization ( Azzolin et al, 2014 ) (Module Box V and Figure 4C ). The third is to act through niche mechanics- and ROS-interdependent integrin signaling ( Werner and Werb, 2002 ; Gregg et al, 2004 ; Buricchi et al, 2007 ; Taddei et al, 2007 ; Zeller et al, 2013 ; Xu Z. et al, 2021 ), where the activation of ILK potentiates Notch signaling ( Maydan et al, 2010 ) and regulates GSK3β activity ( Maydan et al, 2010 ).…”