In previous views the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. However, research of the last decades has revealed that nucleotides also occur in the extracellular space. This survey deals with the sources, metabolism and the role in blood of the extracellular adenine mononucleotides ATP, ADP, AMP and the dinucleotides diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). The latter two are novel compounds, which have recently been discovered in human platelets. The mononucleotides originate from damaged tissues, from red blood cells during haemolysis, from activated platelets, the working muscle and from the nervous system, whereas the dinucleotides are exclusively released from stimulated platelets. Both the adenine mono- and the dinucleotides act as signal molecules on blood cells as well as on cells of the vascular wall, thereby modulating physiological processes such as platelet aggregation, histamine release from mast cells, regulation of vascular tone and white cell functions. In order to limit the signal effects of extracellular nucleotides, blood cells, plasma and the interior of the vessel walls are provided with nucleotide splitting enzymes: ATP, ADP and AMP are mainly degraded by ectoenzymes present on blood cells, endothelial and on smooth muscle cells, whereas dinucleotides are primarily metabolized by plasma enzymes. This review closes with the presentation of the clinical utility of Ap3A and Ap4A as tools for the diagnosis of platelet storage pool defects.
The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.
Adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets. After activation of the platelets both dinucleotides are released into the extracellular milieu where they play a role in the modulation of platelet aggregation and also in the regulation of the vasotone. It has recently been shown that the dinucleotides are degraded by enzymes present in the plasma [Luthje, J. & Ogilvie, A. (1987) Eur. J . Biochem. 169, 385-3881. The further metabolism as well as the role of blood cells has not been established. The dinucleotides were first degraded by plasma phosphodiesterases yielding ATP (ADP) plus AMP as products which were then metabolized to adenosine and inosine. The nucleosides did not accumulate but were very rapidly salvaged by erythrocytes yielding intracellular ATP as the main product.Although lysates of platelets, leucocytes and red blood cells contained large amounts of Ap3A-degrading and Ap4A-degrading activities, these activities were not detectable in suspensions of intact cells suggesting the lack of dinucleotide-hydrolyzing ectoenzymes. Compared to ATP, which is rapidly degraded by ectoenzymes present on blood cells, the half-life of Ap4A was two to three times longer.Since the dinucleotides are secreted together with ADP and ATP from the platelets, we tested the influence of ATP on the rate of degradation of Ap4A. ATP at concentrations present during platelet aggregation strongly inhibited the degradation of Ap,A in whole blood. It is suggested that in vivo the dinucleotides are protected from degradation immediately after their release. They may thus survive for rather long times and may act as signals even at sites far away from the platelet aggregate.Adenosine(5')tetraphospho(5')adenosine (Ap,A) and the homologue adenosine( 5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets [l, 21. Both dinucleotides are released from the platelet dense granules into the extracellular milieu after activation of the cells [l, 21. Ap3A and Ap4A have been suggested to play a role in platelet physiology. Ap4A inhibits ADP-induced platelet aggregation in platelet-rich plasma as well as in whole blood [3, 41. In contrast, Ap3A causes a gradual aggregation of platelets. This Ap3A-induced aggregation is mediated by an enzymatic activity in plasma that hydrolyzes Ap3A, producing ADP, a potent stimulator of platelet aggregation [4]. Furthermore, previous results have shown that Ap4A and Ap3A not only modulate platelet aggregation but also have vasomotor effects [5]. Thus, increasing experimental evidence suggests that these dinucleotides are regulatory molecules involved in the complex process of hemostasis.With respect to their possible role as extracellular signals it is of interest to know how long the dinucleotides can survive Enzymes. The enzymes in the plasma splitting Ap,A and Ap4A are 5'-nucleotide phosphodiesterases (EC 3.1.4.1); lactate dehydrogenase (EC 1.1.1.27).in the blood stream. After their releas...
A hydrolase splitting adenosine(S)triphospho(S')adenosine (Ap3A) to AMP and ADP has recently been detected in human plasma [Liithje, J. and Ogilvie, A. (1984) Biochem. Biophys. Res. Commun. 118,. The enzyme has been purified to apparent homogeneity, as stained in a native polyacrylamide gel. From gel filtration data a Stokes radius of 5.9 nm was calculated, suggesting a molecular mass of about 230 kDa. The presence of the non-ionic detergent Triton X-100 did not change the molecular mass. The hydrolase dissociated to three major protein components (66 kDa; 45 kDa; 16 kDa) during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. Binding of the native enzyme to concanavalin-ASepharose and specific inhibition of binding by methyl mannoside indicated that the hydrolase is a glycoprotein. Two of the subunits (66 kDa; 45 kDa) could be affinity-labeled with radioiodinated concanavalin A.Active hydrolase could be prepared in buffers without added metal ions. Treatment with EDTA, however, completely abolished the hydrolytic activity. The enzyme could be reactivated by incubation with Ca2 + , Co2 + and, at best, with Zn2+, whereas Mg2+ was ineffective. The affinity of the enzyme for Ap3A was high (K, = 1 pM), with normal Michaelis-Menten kinetics. The homolog dinucleotide Ap4A was also substrate (K, =0.6 pM) yielding AMP and ATP as products after the asymmetric split. Other dinucleotides, such as NAD, and also mononucleotides (ATP, UTP) were degraded to nucleoside monophosphates indicating a broad specificity of the enzyme. The synthetic compound thymidine 5'-monophosphate p-nitrophenyl ester was substrate with low affinity whereas its 3'-homolog was not hydrolyzed. Optimal activity of the hydrolase was found at pH 8.5. Adenosine(5')triphospho(5')adenosine (Ap3A) has recently been identified in eukaryotic cells [l, 21. It has been suggested that Ap3A might play an antagonistic role toward the homolog dinucleotide Ap4A, which is a potential signal molecule for the initiation of DNA replication [3-51 and might pleiotropically act as an intracellular 'alarmone' [6, 71. Human platelets store Ap3A as well as Ap4A in a metabolically inactive state [2, 81. Both dinucleotides are released on exposure to thrombin, which induces platelet activation. When added to platelet-rich plasma, Ap3A gradually induces aggregation of the platelets [9]. The addition of Ap4A antagonizes this effect by rapidly disaggregating the platelets. These studies suggested that both dinucleotides also have an extracellular function in modulating platelet aggregation and, therefore, in regulating the initiation of hemostasis.The molecular mechanism of aggregation induced by Ap,A has been clarified [9] by showing a slow degradation of Abbreviations. Ap3A, adenosine(5')triphospho(5')adenosine; Ap,A, adenosine(5')tetraphospho(5')adenosine; SDS, sodium dodecyl sulfate.
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