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* This work was supported by Sandoz Pharma, Quebec Heart and Stroke Foundation, and the Natural Sciences and Engineering Research Council of Canada. This is manuscript no. 695 from our laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) S73813. ¶ Recipient of a studentship award from Fonds pour la Formation de Chercheurs et l'Aide a la Recherche du Quebec and from the Heart and Stroke Foundation of Canada.
Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLGGASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.
The quiescent vascular endothelium maintains blood fluidity by inhibiting blood clotting, by the regulation of platelet activation and adhesion, and by promoting fibrinolysis. In large part, the antithrombotic effects are mediated by surface molecules present on resting endothelial cells (EC),
). ECs also have the potential to regulate platelet activation by the synthesis of prostacyclin and nitric oxide and by the surface expression of ATP diphosphohydrolase (ATPDase). This ecto-enzyme (EC 188.8.131.52) has been also described as apyrase, ecto-ATPase, ecto-ADPase, nucleotide phosphohydrolase, or ATP pyrophosphohydrolase (
). ATPDases are generally low abundance proteins that have been difficult to purify, given their sensitivity to detergents and the propensity to co-isolate with other proteins. High levels of ATPDase activity are, however, associated with the vasculature (endothelium, smooth muscle, and cardiac cells), lymphocytes, and platelets (
). We have recently provided evidence for the loss of ATPDase activity following EC activation, such as would occur in vascular inflammatory states or xenograft rejection, where platelet deposition is a consistent component (
). Such changes in the level of expression and activity of the ATPDase may, therefore, be of pathogenetic significance and prompted our attempts to identify and study this vascular ecto-enzyme.
The biochemical characterization of ATPDase purified from various tissues and organisms has generally revealed a highly glycosylated protein of molecular mass 70-100 kDa. This ecto-enzyme is Ca2+- and Mg2+-dependent, is not sensitive to known inhibitors of the various other ATPases, and hydrolyzes nucleoside tri- and diphosphates but not monophosphates. These enzymatic characteristics have facilitated classification of these proteins into a subgrouping termed E-type ATPases (
). CD39 is known to be an acidic glycoprotein with molecular mass 70-100 kDa that contains two potential transmembrane regions and six potential glycosylation sites. Interestingly, CD39 was originally described as a B-cell activation marker and has been shown to be expressed on the surface of other activated lymphocytes and quiescent vascular endothelium (
). Additionally, CD39 is considered to participate in the enhancement of cell-cell interactions; monoclonal antibodies (mAb) to certain epitopes of CD39 induce homotypic adhesion, probably through involvement of LFA-1 (CD11a/CD18) (
). Potato apyrase (a plant ATPDase) has been recently purified and was independently found to have sequence homology to certain newly identified nucleotide triphosphatases and to murine and human CD39 (
). Because the functional significance of these observations remains unclear, we have further evaluated the role of CD39/ATPDase in modulating platelet reactivity and examined the distribution and nature of CD39 mRNA expression in human tissues.
Our data demonstrate in a persuasive manner that CD39 encodes the vascular ATPDase. This conclusion was originally based on the discovery of sequence homologies between CD39 and human placental ATPDase and bovine aortic ATPDase (Fig. 1). Our hypothesis was further substantiated by other recognized sequence homologies that we noted in mammalian and avian nonvascular ecto-enzymes and the report that the potato soluble apyrase contains certain “apyrase conserved regions” also found in garden pea nucleoside triphosphatase, Saccharomyces cerevisiae golgi guanosine diphosphatase (GDPase), Toxoplasma gondii isoforms of an nucleoside triphosphatase, NTP1 and NTP3, a yeast hypothetical 71.9-kDa protein, a Caenorhabditis elegans 61.3-kDa protein, and human and murine CD39 (
). The following observations also strengthen our conclusion. Both ATPDases and CD39 are known to be membrane glycoproteins with the same molecular weight range. Both proteins have a comparable cellular distribution, are postulated to be involved in cell adhesion, and may undergo up-regulation after viral transformation of certain cells (
We were able to generate CD39 cDNA from human umbilical endothelial cells RNA by reverse transcription-PCR. This PCR product, which was of expected size, was then subjected to restriction mapping and sequencing, which confirmed that this product represented true CD39 cDNA (data not shown). The CD39 cDNA was then cloned into the pCDNA3 vector and expressed in COS-7 cells. Using cell membranes or whole-cell lysates, we established that CD39 protein expressed by these transiently transfected cells reacted with both monoclonal antibodies to CD39 and to polyclonal antibodies directed at ATPDase. By FACS analysis using mAb to CD39, we clearly demonstrated that ATPDase was expressed at the surface of COS-7 cells (Fig. 2). Both polyclonal antibody generated by us to the porcine ATPDase N-terminal peptide fragment and cross-reactive with the bovine vascular ATPDase and monoclonal antibody to CD39 detected the appropriate and same mobility band on Western blotting (Fig. 3).
Our functional data show for the first time that the ATPDase activity associated with CD39 expressed by COS-7 cells can hydrolyze the substrate ADP. This specific ADPase enzyme activity can be induced over 100-fold by the COS-7 transfection with CD39. Likewise, we were able to show enzymatic activity of CD39 for the substrate ATP to be induced over 30-fold by COS-7 transfection (394 nmol/mg protein/min in representative experiments). We also have demonstrated that intact transfected COS-7 cells were able to hydrolyze radiolabeled ADP. This latter result obtained by TLC clearly indicated that the active site of ATPDase faces the extracellular milieu. Wang and Guidotti (
) have also established that Epstein-Barr virus-transformed B cells express both CD39 and potentially Ca2+, Mg2+ apyrase activity. They were able to show by the DEAE-dextran method that COS-7 cells developed 5.4-fold increased ecto-ATPase activity following transfection with CD39 cDNA prepared from B cells, when compared to vector alone (
Of potential significance was our observation that preparations of cell membranes from COS-7 cells transfected with pCDNA3-CD39 could inhibit platelet aggregation in response to ADP, collagen, and thrombin (Fig. 5). ADP release from platelet granules is a vital part of the feedback process that amplifies and propagates platelet activation induced by ADP itself or other more potent agonists (
). Hence, ADP may be an important mediator of vascular thrombosis in inflammatory states. It has been suggested that a major role for the ATPDase may be to inhibit ADP or ATP-induced signal transduction in platelets, leukocytes, and vascular endothelium mediated through the purinergic receptors P2t and P2y (
). It is further possible that the interaction of adenosine nucleotides with CD39 may also influence cell signaling and integrin affinity for their respective ligands. The identification of CD39 as the vascular ATPDase and the documentation of the significant role in modulating platelet reactivity in vitro will further help to test this hypothesis in several experimental models.
Our observation that exposure of COS-7 CD39 transfectants to reactive oxygen intermediates results in loss of platelet antiaggregatory properties coupled to inhibition of biochemical ATPDase activity is in keeping with the oxidant-dependent loss of ATPDase function noted in association with EC activation in vitro and following vascular injury in vivo (
). We speculate that this loss, and the resultant decreased capacity to degrade ADP, could play a significant role in the extensive platelet activation and vascular inflammation seen in graft rejection and other forms of vascular injury. Certainly, the intravenous administration of apyrases to experimental animals has been shown to prolong xenograft survival and abrogate the platelet activation and deposition seen in this setting (
Because of the variable sensitivity of organs to vascular injury and thrombosis, the tissue-specific distribution for CD39/ATPDase was studied. Northern analysis of RNA extracted from different tissues was, therefore, performed with a full-length CD39 probe (Fig. 7). Among the tissues examined, the strongest signals were observed in certain highly vascularized organs, i.e. placenta, lung, skeletal muscle, and kidney. Heart and liver had lower levels of mRNA transcripts. Two dominant CD39 mRNA transcripts are noted in most tissues akin to the pattern observed when mRNA preparations from cultures of human ECs are studied (data not shown). However, we were also able to detect as many as five different mRNA transcripts reacting with the entire CD39 cDNA probe (Fig. 7) and the 3′ and 5′ cDNA regions. These data suggest that these multiple RNA transcripts probably are alternative splicing variants. The pathophysiological significance of this observation is presently undetermined. Interestingly, there was no convincing evidence for the presence of CD39 transcripts in RNA isolated from human brain. This last observation suggests that the specialized vascular tissues of the brain may not express CD39 at levels comparable to the other organs tested and does not explain the data published previously showing the presence of ATPDase activity in nerve tissues and on the external surface of intact synaptosomes (
). Possibly other E-type ATPases or ATPDases unrelated to CD39 are expressed in brain and hepatobiliary tissues, as would be suggested by the finding that ATPase activity may be demonstrated in immunoprecipitated neural cell adhesion molecule from rat brain (reviewed in Ref.
), and regions sensitive to oxidative reactions by sequential mutagenesis experiments will help elucidate the reason(s) for the potential posttranslational modification or other modulation of ATPDase activity with EC activation. This knowledge should permit us to express CD39/ATPDase in an active form despite EC activation, as we have done for thrombomodulin (
We thank Medical Research Council (South Africa), University of Cape Town (MRC, UCT) Liver Center for support of S. C. R., Dr. H. Winkler for expert advice, Dr. J. K. Blusztajn for discussion, and Dr. S. Grey for assistance with FACS analysis.