Characterization of circulating microparticle-associated CD39 family ecto-nucleotidases in human plasma

Abstract

Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ectonucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound ENTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either −2 or/and −3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.

Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice

“…50 The presence of microparticles has been shown in human plasma, 51 and dysregulation of their activity has been associated with human disorders such as idiopathic pulmonary hypertension. 52 Additional studies are needed to determine if monocyte-derived microparticles modulate arterial thrombosis in the setting of increased CD39 expression.…”

Purinergic signaling during intestinal inflammation

“…We have demonstrated the presence of E-NTPDase activity in circulating MP isolated in human plasma (42). Most importantly, the mRNA within MPs can be taken up by these cells and further translated.…”

Protective effects of coffee consumption following liver transplantation for hepatocellular carcinoma in cirrhosis

“…Next, co‐incubation of each radio‐labelled substrate with different combinations of ectoenzyme inhibitors was employed to understand the enzymes participated in each degradation step (Figure E; inhibitor specificity was listed at the bottom). POM6 markedly inhibited ADO production from ADP, suggesting the involvement of ENTPD3; whereas POM1 also worked with decreased potency.…”