S-Nitrosylation of Platelet α_IIbβ₃ As Revealed by Raman Spectroscopy

Abstract

The exact mechanisms regulating conformational changes in the platelet-specific integrin alphaIIbbeta3 are not fully understood. However, a role exists for thiol/disulfide exchange in integrin conformational changes leading to altered disulfide bonding patterns, via its endogenous thiol isomerase activity. Nitric oxide (NO) accelerates this intrinsic enzymatic activity and, in doing so, reverses the activational state of the integrin on the platelet surface toward a more unactivated one. We propose that it is an S-nitrosylation-induced "shuffling" of thiol/disulfide exchange that regulates this reversal of the activated state of the integrin. In this study, we use Raman spectroscopy to explore S-nitrosylation of purified alphaIIbbeta3. Using S-nitrosoglutathione (GSNO) as a model system, we identify Raman markers which show a direct interaction between NO and the thiol groups of the integrin and reveal many of the structural changes that occur in alphaIIbbeta3 in the course of not only its activation but also its deactivation. Key conformational changes are detected within the integrin when treated with manganese (Mn2+), occurring mainly in the cysteine and disulfide regions of the protein, confirming the importance of thiol/disulfide exchange in integrin activation. These changes are subsequently shown to be reversed in the presence of NO.

Vascular thiol isomerases

“…32 PDI undergoes S-nitrosylation following exposure to NO donors. 33 In contrast to S-nitrosylation at regulatory cysteines within platelet receptors such as a IIb b 3 , 34 S-nitrosylation of catalytic cysteines within PDI enables transfer of NO to other proteins and into cells. Several lines of evidence demonstrate this transnitrosylase activity of PDI.…”

Redox Modulation of Adjacent Thiols in VLA-4 by AS101 Converts Myeloid Leukemia Cells from a Drug-Resistant to Drug-Sensitive State

“…Yet, a differential reactivity of various cysteines toward AS101 may exist, possibly reflecting their variable access to AS101. Indeed, a IIb b 3 is subject to S-nitrosylation at specific critical cysteines residues located within specific motifs, resulting in the integrin loss of function (42). This agent does not, however, deactivate all other types of integrins.…”

Integrins and Nitric Oxide in the Regulation of Glial Cells: Potential Roles in Pathological Pain

“…In platelets, integrin alphaIIb/beta3 has been shown to be S-nitrosylated upon exposure to NO donors. This modification favors an inactive integrin state [105]. In addition to the integrin itself, NO induces protein S-nitrosylation of those cytoplasmic molecules important for integrin functions such as actin [106] and calpain [107].…”

“…Integrins are thought to regulate glial migration to, and contact with, target cells, thereby playing a critical role in positioning glial cells. Cooperation and crosstalk between NO and integrins have been investigated in platelets [105], neutrophils [106], and endothelial cells [108,109], but not in glial cells. Therefore, it is imperative that future studies are carried out in order to address how the interplay between NO and integrins impacts on glial cell functions and on the progression of pathological pain.…”