Differential inhibition of human platelet aggregation and thromboxane A2 formation by L-arginine in vivo and in vitro

Abstract

We compared the effects of L-arginine (L-ARG), the precursor of endogenous NO, on platelet aggregation and thromboxane A₂ formation in vivo and in vitro. Human platelet-rich plasma (PRP) was anticoagulated with citrate (which decreases extracellular Ca²⁺) or with recombinant hirudin (which does not affect extracellular Ca²⁺). Two groups of 10 healthy male volunteers received intravenous infusions of L-ARG (30 g or 6 g, 30 min) or placebo. Blood was collected immediately before and at the end of the infusions for aggregation by ADP or collagen. Infusion of L-ARG inhibited ADP-induced aggregation in PRP anticoagulated with citrate by 37.5 ± 6.3% (P < 0.05). In PRP anticoagulated with hirudin, aggregation was inhibited by 33.6 ± 16.0% (P < 0.05). L-ARG infusion also inhibited platelet TXB₂ formation and slightly, but not significantly decreased the urinary excretion rate of 2,3-dinor-TXB₂; cGMP concentrations in PRP were significantly elevated during L-arginine infusion. In vitro preincubation with L-ARG (10 μM–2.5 mM) inhibited platelet aggregation in PRP anticoagulated with r-hirudin, but not citrate. This effect was stereospecific for L-arginine, as D-arginine had no effect. It was dependent upon NO synthase activity, as indicated by increased cGMP levels in PRP. Moreover, both the NOS inhibitor L-NMMA and the inhibitor of soluble guanylyl cyclase ODQ antagonized the effects of L-ARG. Haemoglobin, an extracellular scavenger of NO, partly antagonized the antiplatelet effects of L-ARG. 8-Br-cyclic GMP and the exogenous NO donor linsidomine inhibited aggregation in PRP anticoagulated with citrate or r-hirudin. The inhibitory effects of L-ARG on platelet aggregation in vitro were paralleled by increased cyclic GMP levels; L-ARG also inhibited platelet TXB₂ formation in PRP anticoagulated with r-hirudin, but not citrate. We conclude that the L-arginine/NO pathway is present in human platelets as a Ca²⁺-dependent anti-aggregatory pathway. In vivo the formation of NO from L-ARG by endothelial cells may contribute to the platelet-inhibitory effects of L-ARG. NO-releasing compounds like linsidomine inhibit platelet aggregation in vitro independent of extracellular Ca²⁺.