Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.
Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.
Martín-Valmaseda
E M
EM
Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Edificio Departamental, Lab. 106, Plaza Doctores de la Reina s/n, E-37007, Salamanca, Spain.
Sánchez-Yagüe
J
J
Rodríguez
M C
MC
Gómez
F P
FP
Llanillo
M
M
D000110:Acetylcholinesterase / Q000737:chemistry; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D016922:Cellular Senescence; D015925:Cryopreservation; D004791:Enzyme Inhibitors / Q000737:chemistry; D005227:Fatty Acids / Q000032:analysis; D005950:Glucose Solution, Hypertonic; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors; D066298:In Vitro Techniques; D015227:Lipid Peroxidation; D008560:Membrane Fluidity; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry; D010949:Plasma; D012756:Sheep; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D013997:Time Factors
D004791:Enzyme Inhibitors; D005227:Fatty Acids; D005950:Glucose Solution, Hypertonic; D010743:Phospholipids; C076429:Seto solution; D017392:Thiobarbituric Acid Reactive Substances; D000110:Acetylcholinesterase; D005952:Glucose-6-Phosphatase