Malondialdehyde formation in rat platelet-rich plasma. I.A kinetic approach

Malondialdehyde (MDA) is a stable produce of arachidonic acid metabolism, catalyzed by the enzyme cyclo-oxygenase. The experimental conditions for measuring the kinetics of MDA formation in rat citrated platelet-rich plasma were defined. Platelets were stimulated with either arachidonic acid, the substrate of MDA, or thrombin, an enzyme which induces release of free arachidonic acid from platelet membrane phospholipids. MDA formation was almost linear for a limited period of time (between 0 and 2 min with arachidonic acid and between 1 and 3 min with thrombin) and was concentration-dependent with saturation kinetics. The hyperbolic curves obtained could be recast in linear plots (according to Woolf transformation S/V versus S) when arachidonic acid was used. With thrombin, in contrast, the highest concentration at which no MDA production could be detected (3 NIH u/ml) had to be subtracted from each concentration of the enzyme used to obtain Woolf plots. The apparent Km value of arachidonic acid was 0.49 /+- 0.09 mM and Vmax was 1.44 /+- 0.06 nmoles MDA/1.4 Z 10(9) platelets/min. The corresponding values in experiments with thrombin were 6.5 /+- 1.5 NIH u/ml and 0.233 /+- 0.012 nmoles MDA/1.4 X 10(9) platelets/min.