Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities

Abstract

Extracellular ATP and ADP trigger inflammatory, vasodilatatory, and prothrombotic signaling events in the vasculature, and their turnover is governed by networks of membrane-associated enzymes. The contribution of soluble activities to intravascular nucleotide homeostasis remains controversial. By using thin-layer chromatographic assays, we revealed transphosphorylation of [γ-(32)P]ATP and AMP by human and murine sera, which was progressively inhibited by specific adenylate kinase (AK) inhibitor Ap(5)A. This phosphotransfer reaction was diminished markedly in serum from knockout mice lacking the major AK isoform, AK1, and in human serum immunodepleted of AK1. We also showed that ∼75% ADP in cell-free serum is metabolized via reversible AK1 reaction 2ADP ↔ ATP + AMP. The generated ATP and AMP are then metabolized through the coupled nucleotide pyrophosphatase/phosphodiesterase and 5'-nucleotidase/CD73 reactions, respectively. Constitutive presence of another nucleotide-converting enzyme, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, known as CD39), was ascertained by the relative deficiency of serum from CD39-null mice to dephosphorylate [(3)H]ADP and [γ-(32)P]ATP, and also by diminished [(3)H]ADP hydrolysis by human serum pretreated with NTPDase1 inhibitors, POM-1 and ARL-67156. In summary, we have identified hitherto unrecognized soluble forms of AK1 and NTPDase1/CD39 that contribute in the active cycling between the principal platelet-recruiting agent ADP and other circulating nucleotides.

Figure 1.

TLC analysis of purine-converting pathways in the serum from AK1^−/− mice. A, B) Serum from AK1^−/− and wild-type (AK^+/+) mice was incubated with 50 μM [γ-³²P]ATP in the absence (vehicle) or presence of 1 mM AMP and UDP (A), as well as with 100 μM [³H]ADP (B). Some samples were pretreated with 80 μM Ap₅A prior to addition of tracer substrates. Blanks (Bl) show the radiochemical purity of tracer substrates in the absence of serum. Arrows indicate the positions of nucleotide standards, adenosine (Ado), hypoxanthine (Hyp), and inosine (Ino), as well as inorganic phosphate (P_i), and pyrophosphate (PP_i). C) Sera from AK1^−/− and AK^+/+ mice were assayed for specific AK, ADPase, 5′-nucleotidase (5-NT), adenosine deaminase (ADA), and NDP kinase (NDPK) activities using saturated concentrations of indicated ³H-labeled and unlabeled substrates (means±se n=10–12). *P < 0.05 vs. wild-type controls.

Figure 2.

TLC analysis of soluble nucleotidase activities in CD39^−/− mice. A, B) Sera from CD39^−/− and wild-type (CD39^+/+) mice were incubated with 50 μM [γ-³²P]ATP (A) or with 100 μM [³H]ADP (B). Some samples were pretreated with EDTA (2 mM), POM-1 (100 μM), or Ap₅A (80 μM) prior to addition of tracer substrates. Blanks (Bl) show the radiochemical purities of [γ-³²P]ATP and [³H]ADP. Arrows indicate the positions of nucleotide and nucleoside standards, inorganic phosphate (P_i) and pyrophosphate (PP_i). C) Rate of [³H]ADP (100 μM) hydrolysis was measured in sera from CD39^+/+ and CD39^−/− mice in the absence (left panel) or presence (middle panel) of 80 μM Ap₅A, while 5′-nucleotidase (5-NT) was assayed using 300 μM [³H]AMP as substrate (right panel). Data are presented as means ± se (n=12). *P < 0.05 vs. wild-type controls.

Figure 3.

Progressive inhibition of soluble AK activity in human and murine blood by Ap₅A. Sera from human volunteers (n=5) and from C57Bl/6 wild-type mice (n=3) were preincubated for 20 min with increasing concentrations of Ap₅A (0–120 μM). Direct and reverse AK activities were determined by the rates of [³H]AMP and ATP transphosphorylation (A) and [³H]ADP conversion into dephosphorylated ³H-metabolites (B), respectively. Results are plotted as the percentage of maximal activity measured in the absence of Ap₅A (defined as 100%), and IC₅₀ values were calculated from the obtained competitive curves using a nonlinear curve-fitting program (means±se).

Figure 4.

Autoradiographic analysis of [³H]ADP metabolism in human blood. Serum and heparinized plasma were collected from venous blood of 2 volunteers. Blood samples (10 μl) were pretreated for 20 min in the absence or presence of 100 μM Ap₅A prior to addition of 50 μM [³H]ADP. After 60 min incubation, aliquots of the reaction mixture were separated by TLC and developed by autoradiography. Blank (Bl) shows the radiochemical purity of [³H]ADP. Arrows indicate the positions of nucleotide standards and adenosine.

Figure 5.

Pattern of nucleotide metabolism in human blood. Purinergic activities were determined in human serum and heparinized plasma, as well as in serum filtrates passed through 0.22-μm filters (n=4). A) Left panel: Soluble AK was assayed by TLC using 400 μM [³H]AMP plus 750 μM ATP. Right panel: human serum was also depleted of AK1 with Protein A Sepharose CL-4B beads to which rabbit anti-AK1 antibodies (AK1) or normal rabbit serum (NRS; serving as irrelevant negative control) had been coupled. AK activity was assayed both in serum supernatants and resuspended bead pellets and expressed as micromoles of [³H]AMP phosphorylated by 1 ml of serum or beads per hour (means±se; n=3). B) Soluble NTPDase/ADPase was assayed by TLC as in A, using 50 μM [³H]ADP in the presence of 100 μM Ap₅A. C) Serum was pretreated with 300 μM POM-1, ARL-67156, and PPADS prior to addition of [³H]ADP. Graph shows soluble [³H]ADP-hydrolyzing activity plotted as the percentage of control nucleotidase activity measured in the absence of inhibitors (means±se; n=6–8). *P < 0.05 vs. corresponding controls.

Figure 6.

Major purine-converting pathways in cell-free serum. Scheme highlights the potential exchange activities of circulating adenine nucleotides and adenosine and additionally outlines further conversion of adenosine-derived inosine into hypoxanthine, xanthine, and uric acid. The following soluble purinergic activities have been identified: nucleotide pyrophosphatase/ phosphodiesterase (NPP), NTPDase1/CD39, 5′-nucleotidase/CD73 (5NT), adenylate kinase-1 (AK1), NDP kinase (NDPK), and adenosine deaminase (ADA).