Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: Kinetic characterization and biochemical properties

Abstract

In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. K_m and V_max values for p-Nph-5′-TMP hydrolysis were found to be 106 ± 18 µM and 3.44 ± 0.18 nmol p-nitrophenol/min/mg (mean ± SD, n = 5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5′-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25 mM suramin inhibited p-Nph-5′-TMP, ATP and ADP hydrolysis, while 0.5 mM AMP decreased only p-Nph-5′-TMP hydrolysis. Besides, 5.0, 10 and 20 mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5 mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.

• Platelets
• nucleotide hydrolysis
• E-NPP
• PDEase
• E-NTPDase