Original Articles
Role of extracellular ATP metabolism in regulation of platelet reactivity,☆☆

https://doi.org/10.1067/mlc.2002.126719Get rights and content

Abstract

Extracellular adenosine triphosphate (ATP) regulates platelet reactivity by way of direct action on platelet purinergic receptors or by hydrolysis to adenosine diphosphate (ADP). Subsequent metabolism of ATP and ADP to adenosine monophosphate (AMP) and adenosine inhibits platelet aggregation. Endothelial cell membrane-bound ecto-ATP/ADPase (CD39, E-NTPDase1) is thought to be the main regulator of platelet responsiveness. However, the findings in studies of CD39-knockout mice imply that nucleotidase(s) in plasma regulates circulating adenine nucleotides levels. Understanding extracellular ATP metabolism by CD39 and plasma nucleotidases is therefore important. In this study, α-phosphorus 32- and γ-phosphorus 32-labeled ATP were rapidly metabolized directly to AMP and pyrophosphate in human plasma at pH 7.4, suggesting the presence of pyrophosphatase/phosphodiesterase-like activity. A specific phosphodiesterase substrate, p-nitrophenol-5′-TMP (p-Nph-5′-TMP), was readily hydrolyzed in human plasma. The antiaggregatory action of β,γ-methylene-ATP (AMPPCP) (5 μmol/L) was blocked by DMPX, an adenosine-receptor antagonist, suggesting that in plasma, AMPPCP was metabolized to AMP and adenosine. Recombinant soluble CD39 (solCD39) was used to assess the role of CD39 in ATP metabolism. As little as 0.25 μg/mL of solCD39 inhibited ADP-induced platelet aggregation. However, in the presence of ADP-free ATP (10 μmol/L), solCD39 induced platelet aggregation in a dose-dependent manner. Because AMPPCP could not substitute for ATP in solCD39-stimulated platelet aggregation, it is likely that ADP formation from ATP was required. Endogenous CD39 may thus have a hemostatic function by promoting ADP formation from released ATP, in addition to its antiaggregatory properties. A plasma nucleotidase hydrolyzes ATP directly to AMP. This prevents ADP accumulation and generates adenosine, a potent, locally acting inhibitor of platelet reactivity. The presence of both endothelial CD39 and plasma nucleotidase appears to be important in the maintenance of normal hemostasis and prevention of excessive platelet responsiveness. (J Lab Clin Med 2002;140:166-75)

Section snippets

Chemicals and reagents

Recombinant solCD39 was prepared as described previously.16 α-[32P]ATP and γ-[32P]ATP (3000 Ci/mmol, 10 mCi/mL of 50 mmol/L Tricine, pH 7.6) were obtained from NEN Life Science Products (Boston, Mass). α-[32P]ADP was prepared from α-[32P]ATP by means of enzymatic degradation with alkaline phosphatase (Promega, Madison, Wis) and separated with the use of paper electrophoresis on DEAE paper at pH 1.9, followed by elution.27 All other chemicals and reagents were obtained from Sigma Chemical Co (St

Results

ATP alone had no effect on platelet aggregation, even when doses as high as 50 μmol/L were added to human PRP (Fig 1).

. Effects of ATP and ADP on human platelet aggregation. Although ATP (50 μmol/L) did not cause platelet aggregation, it inhibited platelet aggregation induced by 1 μmol/L ADP. All aggregation experiments were carried out for 4 minutes. Arrows indicate the addition of various compounds. Tracings are representative of three independent experiments that yielded similar results.

Discussion

Control of circulating nucleotide levels is important in the maintenance of physiological nucleotide-mediated signaling processes. Such control is provided in part by endothelial cell CD39, which, in concert with prostacyclin and nitric oxide, primarily accounts for maintenance of blood fluidity.28, 29 Our results show that circulating ADP and ATP also can be efficiently hydrolyzed by a soluble enzyme in human plasma. In plasma, ATP was hydrolyzed directly to AMP without any significant

References (38)

  • AD Branch et al.

    RNA fingerprinting

    Methods Enzymol

    (1989)
  • W Chen et al.

    Soluble apyrases release ADP during ATP hydrolysis

    Biochem Biophys Res Commun

    (2001)
  • HS Park et al.

    Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adnosine 5-diphosphate (ADP) in human platelets

    Br J Pharmacol

    (1999)
  • MG Rolf et al.

    Platelet shape change evoked by selective activation of P2X1 purinoceptors with alpha,beta-methylene ATP

    Thromb Haemost

    (2001)
  • C Oury et al.

    The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen

    Thromb Haemost

    (2001)
  • AJ Marcus et al.

    Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase: thromboregulation by endothelial cells

    J Clin Invest

    (1991)
  • AJ Marcus et al.

    The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39

    J Clin Invest

    (1997)
  • H. Zimmermann

    Extracellular metabolism of ATP and other nucleotides

    Naunyn Schmiedebergs Arch Pharmacol

    (2000)
  • CR Maliszewski et al.

    The CD39 lymphoid cell activation antigen: molecular cloning and structural characterization

    J Immunol

    (1994)
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    Supported in part by National Institutes of Health grants R37-DA02475 (HHS), DK56424-02 (HDR), HL-46403 (AJM, MJB), HL-47073 (AJM, MJB), and NS-41462 (AJM, MJB); predoctoral fellowship F31-DA05862-01 from the National Institute on Drug Abuse (AVB); NIDA training grant DAO7274 from the National Institutes of Health (AVB); and merit review grants from the Department of Veterans Affairs (AJM, MJB, JHFD). We thank Charles R. Maliszewski, PhD, and colleagues (Immunex Corp, Seattle, Wash) for a generous gift of solCD39.

    ☆☆

    Reprint requests: Hazel H. Szeto, MD, PhD, Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021; e-mail: [email protected].

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