Nitric oxide inhibits von Willebrand factor-mediated platelet adhesion and spreading through regulation of integrin alpha(IIb)beta(3) and myosin light chain.

ABSTRACT

BACKGROUND: von Willebrand factor (VWF)-mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors glycoprotein Ib (GPIb)-V-IX and integrin alpha(IIb)beta(3). However, little is known about the negative regulation of these events. OBJECTIVES: To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. RESULTS: S-nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin alpha(IIb)beta(3) demonstrated that NO targeted alpha(IIb)beta(3)-mediated aggregation and spreading, but did not significantly influence GPIb-mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of alpha(IIb)beta(3) on adherent platelets. NO reduced the phosphorylation of extracellular stimuli-responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated alpha(IIb)beta(3)-specific antibody PAC-1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin alpha(IIb)beta(3) and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. CONCLUSION: These data demonstrate that initial activation-independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb-mediated activation of alpha(IIb)beta(3) and MLC leading to reduced platelet spreading and aggregation.