S-Nitrosylation of platelet alphaIIbbeta3 as revealed by Raman spectroscopy.
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.
Walsh
Geraldine M
GM
Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
Leane
Deirdre
D
Moran
Niamh
N
Keyes
Tia E
TE
Forster
Robert J
RJ
Kenny
Dermot
D
O'Neill
Sarah
S
D000818:Animals; D004220:Disulfides; D006801:Humans; D016023:Integrins; D008345:Manganese; D009569:Nitric Oxide; D009589:Nitrogen Oxides / Q000378:metabolism*; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D026422:S-Nitrosoglutathione; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013438:Sulfhydryl Compounds
D004220:Disulfides; D016023:Integrins; D009589:Nitrogen Oxides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D008345:Manganese; D026422:S-Nitrosoglutathione