Extracellular di- and tri-phosphate nucleotides are released from activated or injured cells to trigger vascular and immune P purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ecto-nucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the mono-phosphate nucleoside end-products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ecto-enzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ecto-enzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional e... More
Extracellular di- and tri-phosphate nucleotides are released from activated or injured cells to trigger vascular and immune P purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ecto-nucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the mono-phosphate nucleoside end-products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ecto-enzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ecto-enzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase (ALP-ECD) and acid phosphatase (HAP-ECD) fusion proteins were also generated, characterized and compared with these CD39-ECD, CD73-ECD and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ecto-enzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting tri- and di-phosphate nucleotides into nucleosides when compared to ALP-ECD and HAP-ECD. We also note a pH-sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ecto-enzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge pro-inflammatory ATP and also generate anti-inflammatory adenosine.
