Red blood cell stimulation of platelet nitric oxide production indicated by quantitative monitoring of the communication between cells in the bloodstream

ATP is a recognized stimulus of nitric oxide synthase and is released from red blood cells (RBCs) upon deformation. The objective of this work is to demonstrate that RBCs stimulate nitric oxide production in platelets by employing a continuous flow analysis system in which the stream contains both RBCs and platelets. Here, two drugs known to improve blood flow in vivo (pentoxyfilline and iloprost) are shown to increase both the release of RBC-derived ATP and the production of platelet-derived NO. A flow-based chemiluminescence assay (in vitro) was employed to quantitatively determine the amount of ATP released from erythrocytes subjected to flow-induced deformation. Prior to being subjected to flow, erythrocytes were incubated in the absence or presence of 4.8 mu M pentoxyfilline or 80 nM iloprost. Erythrocytes obtained from rabbits (n = 22) that were subjected to flow released 239 +/- 29 nM ATP. When treated with pentoxyfilline, the ATP released from the flowing RBCs increased to 450 +/- 94 nM ATP. An increase in RBC-derived ATP was also measured for iloprost-incubated RBCs in flow (362 +/- 45 nM ATP). Importantly, platelets that were loaded with diaminofluorofluorescein diacetate, an intracellular fluorescence probe for NO, exhibited increases in fluorescence intensity by 16% in the presence of RBCs treated with pentoxyfilline and a 10% increase when treated with iloprost. When the ATP release from the RBCs was inhibited with glybenclamide, the platelet fluorescence intensity decreased by 25 and 51% for RBCs incubated with pentoxyfilline and iloprost, respectively. In an experiment not involving the RBC, inhibition of the P2x receptor on the platelets (an ATP receptor) resulted in no increase in platelet NO production, suggesting that the NO production in the activated platelet is due to ATP.