Inhibition of human platelet reactivity by endothelium-derived relaxing factor from human umbilical vein endothelial cells in suspension: blockade of aggregation and secretion by an aspirin-insensitive mechanism.
To determine a role for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) in regulation of human platelet reactivity by human endothelial cells (EC), we studied combined suspensions of human umbilical vein endothelial cells (HU-VEC, passage 2 through 3) and washed human platelets. Confluent HUVEC monolayers were treated with aspirin (1 mmol/L) to prevent prostacyclin (PGI2) formation, washed, and harvested. Aspirin-treated platelets alone (58 x 10(6)) were fully aggregated by thrombin at 0.05 U/mL or more. In the presence of 10(6) HUVEC, however, platelet serotonin release and aggregation in response to thrombin at doses as high as 0.5 U/mL were blocked. We demonstrated for the first time that inhibition of aggregation and serotonin release, due to EDRF/NO, occurred in parallel. HUVEC-dependent inhibition of platelet responsiveness was enhanced by superoxide dismutase (SOD) and reversed by hemoglobin. The inhibitory effect was also reversed by preincubation of HUVEC with NG-monomethyl-L-arginine (NMA) or NG-nitro-L-arginine (NNA) through competitive blockade of arginine metabolism. Pretreatment of platelets with methylene blue indicated that EC-dependent inhibition of platelet reactivity occurred through activation of platelet soluble guanylate cyclase. When platelets and HUVEC were separated by a permeable membrane and both cells were stimulated by thrombin, platelets remained unresponsive. This indicated that inhibition was induced by a fluid-phase mediator, independent of direct cell-cell contact. These data demonstrate that EDRF/NO formation from L-arginine by human EC plays an important role as an aspirin-insensitive fluid-phase inhibitor of human platelet reactivity.
Broekman
M J
MJ
Department of Medicine, Department of Veterans Affairs Medical Center, New York, NY.
Eiroa
A M
AM
Marcus
A J
AJ
D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects; D012701:Serotonin / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins; D019323:omega-N-Methylarginine
D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D012701:Serotonin; D001120:Arginine; D013482:Superoxide Dismutase; D013917:Thrombin; D001241:Aspirin