Role of a novel soluble nucleotide phospho-hydrolase from sheep plasma in inhibition of platelet reactivity: Hemostasis, thrombosis, and vascular biology

Abstract

Ecto- and exoenzymes that metabolize extracellular adenosine diphosphate (ADP), the major promoter of platelet activation and recruitment, are of potential clinical importance because they can metabolically prevent excessive thrombus growth. An ecto-ADPase (CD39, NTPDase1) has been identified on endothelial cells. We demonstrate that ADP and adenosine triphosphate (ATP) are rapidly metabolized to adenosine monophosphate (AMP) in sheep plasma at pH 7.4. This hydrolysis is sensitive to P1, P5-di-(adenosine-5′) pentaphosphate (Ap5A), and ethylene glycol bis (β-aminoethyl ether) - N, N, N-, N- tetra-acetate (EGTA) but insensitive to tetramisole (an alkaline phosphatase inhibitor). A specific phosphodiesterase substrate, p -nitrophenol-5′-thymidine monophosphate (TMP) (p -Nph-5′-TMP), was readily hydrolyzed in sheep plasma at a rate of ~0.25 nmol/min/mg protein, and this hydrolysis was inhibited by ADP, ATP, and Ap5A. Furthermore, 200-fold purified p -Nph-5′-TMP-hydrolyzing activity also hydrolyzed ATP and ADP directly to AMP. When ADP was preincubated in plasma, its ability to induce platelet aggregation was inhibited in a time-dependent manner. This effect was abolished by Ap5A. The inhibitory effects on platelet aggregation correlated with hydrolysis of the ADP in plasma. These data suggest that the endogenous soluble plasma phosphohydrolase metabolizes ATP and ADP by means of cleavage of the α-β-phosphodiester bond of nucleoside 5′-phosphate derivatives. This novel biochemical activity inhibits platelet reactivity through hydrolysis of extracellular nucleotides released by activated platelets during (patho)physiological processes, serving a homeostatic and antithrombotic function in vivo. (J Lab Clin Med 2002;139:116-24)