A Kinetic Study of Platelet Malondialdehyde (MDA) Formation and its Pharmacological Inhibition in rat Platelet-Rich Plasma (PRP)

Abstract

Experimental conditions were defined for kinetic analysis of MDA formation and inhibition in PRP prepared from citrated blood of male CD rats. Portions of PRP were stirred at 37°C with different concentrations of thrombin or arachidonic acid (AA) and the reaction was stopped by TCA. The rate of MDA generation (measured by a thiobarbiturate reaction) increased linearly during the first few min, reaching a plateau within 5 min. In all subsequent experiments PRP was incubated with thrombin for 2 ½ min or AA for 2 min. Km for thrombin was about 10 NIH U/ml and for AA 0.35 mM. Vmax were 0.27 and 0.40 nmol. MDA/1.4 × 109 platelets/min. Acetylsalicylic acid (ASA) or indomethacin (I) were incubated with PRP at 37°C for either 1 or 10 min before addition of thrombin or AA. Both drugs inhibited MDA formation in an apparently competitive way. Ki values were 10-30 times lower for I than for ASA. Ki values Of both drugs were about 20 times lower after 10 than after 1 min incubation. The inhibitory effect of either drug could be decreased by diluting the PRP samples, and appeared to be additive. These data suggest that: 1) rat platelets kept in their own plasma are a suitable model for studying the kinetics of MDA generation; 2) in rat platelets ASA and I act on the same enzymatic intracellular mechanism; 3) irreversible acetylation of platelet cyclo-oxygenase does not account for ASA’s initial inhibitory effect on platelet MDA generation.