Oxidative response and membrane modification of diabetic platelets challenged with PAF.
Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.
Tiano
L
L
Department of M.C.A. Biology, University of Camerino, Italy. l.tiano@cambio.unicam.it
Kantar
A
A
Falcioni
G
G
Littarru
G P
GP
Cherubini
V
V
Fiorini
R
R
D000166:Acridines; D000293:Adolescent; D001792:Blood Platelets / Q000502:physiology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000502:physiology*; D002648:Child; D004161:Diphenylhexatriene / Q000031:analogs & derivatives*; D005260:Female; D005454:Fluorescence Polarization; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood; D007700:Kinetics; D008163:Luminescent Measurements; D008165:Luminol; D008297:Male; D008560:Membrane Fluidity; D010972:Platelet Activating Factor; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000097:blood; D013481:Superoxides / Q000097:blood
D000166:Acridines; D010972:Platelet Activating Factor; D017382:Reactive Oxygen Species; D013481:Superoxides; D004161:Diphenylhexatriene; C033472:10,10'-dimethyl-9,9'-biacridinium; D008165:Luminol; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase