Red Blood Cell Stimulation of Platelet Nitric Oxide Production Indicated by Quantitative Monitoring of the Communication between Cells in the Bloodstream

Abstract

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 flowbased 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 µM 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.In addition to its well-known ability as a vasodilator, NO is also able to inhibit platelet activation and aggregation. Platelets have also been shown to produce NO upon activation. 1 Freedman et al. simultaneously measured NO production and aggregation of platelets upon activation with ATP. 2 These authors placed an electrode for NO into the cell of an aggregometer in order to simultaneously monitor the NO release from platelets and the aggregation of the platelets, thus demonstrating that NO was produced and released by these cells upon activation. From these studies, the authors concluded that NO released by the platelets was key to preventing further platelet recruitment to the activated platelets, but only mildly prevented their adhesion to an endothelium.In question concerning the production and release of plateletderived NO is the mechanism by which the NO production is stimulated. For example, ATP is a recognized stimulus of nitric oxide synthase (NOS) and subsequent production of platelet NO; moreover, it is also known that ATP is released from platelets upon activation. 3 However, it has not been established if ATP released from activated platelets has the ability to stimulate the production of NO in platelets. Here, res...

Effect of Blood Hematocrit and Erythrocyte Deformability on Adenosine 5′-Diphosphate Platelet Reactivity in Patients With Acute Coronary Syndromes on Dual Antiplatelet Therapy

“…23 Moreover, it has been demonstrated that erythrocytes are capable of releasing adenosine triphosphate by mechanical deformation or pharmacologically, thus favoring nitric oxide release by activated platelets. 24 In contrast, anemic patients with ACSs, except those with very low hematocrit, do not seem to benefit from administration of blood transfusions that, on the contrary, are associated with a worse outcome 25 ; the negative impact of blood transfusions in patients presenting with anemia and suspected ACS has been confirmed and ascribed to the change in shape and deformability of stored erythrocytes, 25 which probably release a larger amount of ADP, thus further decreasing platelet responsiveness to antiplatelet drugs.…”

Metabolomics in diabetic complications

“…Oxidative stress has also been shown to inhibit the release of nitric oxide-stimulating ATP from erythrocytes, further contributing to vascular dysfunction. 5,6 …”

Extracellular ATP in the Immune System: More Than Just a "Danger Signal"

“…The ATP released by deformed RBCs may help these cells to communicate not only with endothelial cells but also with other neighbors, such as platelets. Platelets that sense a local increase in [eATP] (due to RBCs or other platelets) also produce NO (19), and this is key to preventing the further recruitment of other platelets to the activated platelets and thus averting obstruction of narrow vessels. A third type of cell-to-cell communication is that which occurs when [eATP] is strongly increased, for example, as happens after cell damage.…”