Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.
We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10-50 microg/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE x DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE x DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE x DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r = 0.85; p < 0.01, n = 10), whereas cyclohexanedione-apoE x DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of L-arginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (100 nM), had a similar reversing action. Direct confirmation that apoE stimulates NO synthase was obtained by use of L-[3H]arginine; platelets pretreated with apoE x DMPC produced markedly more L-[3H]citrulline (0.71 +/- 0.1 pmol/h/10(9) platelets) than controls (0.18 +/- 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway.
Riddell
D R
DR
University Department of Medicine, Royal Free Hospital School of Medicine, London, United Kingdom.
Graham
A
A
Owen
J S
JS
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D001057:Apolipoproteins E / Q000494:pharmacology*; D001120:Arginine / Q000502:physiology*; D001792:Blood Platelets / Q000201:enzymology; D002784:Cholesterol / Q000378:metabolism; D000242:Cyclic AMP / Q000097:blood; D006152:Cyclic GMP / Q000097:blood; D004134:Dimyristoylphosphatidylcholine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D008075:Lipoproteins, HDL / Q000494:pharmacology; D008563:Membrane Lipids / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000097:blood; D010069:Oxadiazoles / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors; D011810:Quinoxalines / Q000494:pharmacology
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D001057:Apolipoproteins E; D004791:Enzyme Inhibitors; D008075:Lipoproteins, HDL; D008563:Membrane Lipids; D010069:Oxadiazoles; D010726:Phosphodiesterase Inhibitors; D010975:Platelet Aggregation Inhibitors; D011810:Quinoxalines; D009569:Nitric Oxide; D001120:Arginine; D002784:Cholesterol; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine; D004134:Dimyristoylphosphatidylcholine