Biomedical subjects

J Sévigny

Publications and source records attributed to J Sévigny.

At least 19 recordsLinked to original sources

PubMed2006

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes.

Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.

Adenosine Diphosphate↗

PubMed2004

Noise exposure induces up-regulation of ecto-nucleoside triphosphate diphosphohydrolases 1 and 2 in rat cochlea.

Extracellular ATP acting via P2 receptors in the inner ear initiates a variety of signaling pathways that may be involved in noise-induced cochlear injury. Nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39 and NTPDase2/CD39L1 are key elements for regulation of extracellular nucleotide concentrations and P2 receptor signaling in the cochlea. This study characterized the effect of noise exposure on regulation of NTPDase1 and NTPDase2 expression in the cochlea using a combination of real-time RT-PCR, immunohistochemistry and functional studies. Adult Wistar rats were exposed to broad band noise at 90 dB and 110 dB sound pressure level (SPL) for 72 h. Exposure to 90 dB SPL induced a small and temporary change of auditory thresholds (temporary threshold shift), while exposure to 110 dB SPL induced a robust and permanent change of auditory thresholds (permanent threshold shift). NTPDase1 and NTPDase2 mRNA transcripts were upregulated in the cochlea exposed to 110 dB SPL, while mild noise (90 dB SPL) altered only NTPDase1 mRNA expression levels. Changes in NTPDases expression did not correlate with levels of circulating corticosterone, implying that the up-regulation of NTPDases expression was not stress-related. Semi-quantitative immunohistochemistry in the cochlea exposed to 110 dB SPL localized the increased NTPDase1 and NTPDase2 immunostaining in the stria vascularis and up-regulation of NTPDase2 in the intraganglionic spiral bundle. In contrast, NTPDase1 was down-regulated in the cell bodies of the spiral ganglion neurones. Distribution of NTPDases was not altered in the cochlea exposed to 90 dB SPL. Functional studies revealed increased ectonucleotidase activities in the cochlea after exposure to 110 dB SPL, consistent with up-regulation of NTPDases. The changes in NTPDases expression may reflect adaptive response of cochlear tissues to limit ATP signaling during noise exposure.

Acoustic Stimulation↗

PubMed2001

Disordered cellular migration and angiogenesis in cd39-null mice.

BACKGROUND: Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 is the major ectonucleotidase of endothelial cells and monocytes and catalyzes phosphohydrolysis of extracellular nucleoside diphosphates (NDP) and triphosphates (NTP, eg, ATP and UTP). Deletion of cd39 causes perturbations in the hydrolysis of NTP and NDP in the vasculature. Activation of P2 receptors appears to influence endothelial cell chemotactic and mitogenic responses in vitro. Therefore, aberrant regulation of nucleotide P2 receptors may influence angiogenesis in cd39-null mice. Methods and Results- In control mice, implanted Matrigel plugs containing growth factors were rapidly populated by monocyte/macrophages, endothelial cells, and pericytes, with the development of new vessels over days. In cd39-null mice, migrating cells were completely confined to the tissue-Matrigel interface in a clearly stratified manner. Absolute failure of new vessel ingrowth was consistently observed in the mutant mice. Linked to these findings, chemotaxis of cd39-null monocyte/macrophages to nucleotides was impaired in vitro. This abnormality was associated with desensitization of nucleotide receptor P2Y-mediated signaling pathways. CONCLUSIONS: Our findings demonstrate a role for NTPDase1 and phosphohydrolysis of extracellular nucleotides in the regulation of the cellular infiltration and new vessel growth in a model of angiogenesis.

Acid Anhydride Hydrolases↗

PubMed2001

P2Y(6) nucleotide receptor mediates monocyte interleukin-8 production in response to UDP or lipopolysaccharide.

Extracellular nucleotides are autocrine and paracrine cellular mediators that signal through P2 nucleotide receptors. Monocytic cells express several P2Y receptors but the role of these G protein-coupled receptors in monocytes is not known. Here, we present evidence that P2Y(6) regulates chemokine production and release in monocytes. We find that UDP, a selective P2Y(6) agonist, stimulates interleukin (IL)-8 release in human THP-1 monocytic cells whereas other nucleotides are relatively inactive. P2 receptor antagonists or P2Y(6) antisense oligonucleotides inhibit IL-8 release induced by UDP. Furthermore, UDP specifically activated IL-8 production in astrocytoma 1321N1 cells transfected with human P2Y(6). Since lipopolysaccharide has been suggested to activate P2 receptors via nucleotide release, we tested whether IL-8 production stimulated by lipopolysaccharide might result from P2Y(6) activation. P2 antagonists or apyrase, an enzyme which hydrolyzes nucleotides including UDP, inhibit IL-8 production induced by lipopolysaccharide but not by other stimuli. Furthermore, IL-8 gene expression activated by lipopolysaccharide is enhanced by P2Y(6) overexpression and inhibited by P2Y(6) antisense oligonucleotides. Thus, UDP activates IL-8 production via P2Y(6) in monocytic cells. Furthermore, lipopolysaccharide mediates IL-8 production at least in part by autocrine P2Y(6) activation. These findings indicate a novel role for P2Y(6) in innate immune defenses.

Cell Line↗

PubMed2001

The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five 'apyrase conserved regions' (ACR1 to ACR5), and a cysteine-rich C-terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N-terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino-acid residues between ACR3 and ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild-type enzymes.

Adenosine Diphosphate↗

PubMed2001

Targeting platelet aggregation: CD39 gene transfer augments nucleoside triphosphate diphosphohydrolase activity in injured rabbit arteries.

BACKGROUND: CD39, the major endothelial nucleoside triphosphate diphosphohydrolase (NTPDase), plays an important role in local thromboregulation. We hypothesized that balloon injury (BI) leads to an acute reduction in arterial NTPDase activity that could be restored by a targeted gene delivery strategy. METHODS: Recombinant adenoviral vectors containing human CD39 (Ad-CD39) or beta-galactosidase (Ad-LacZ) were used. Endothelial (ECs) and smooth muscle cells (SMCs) were infected in vitro and NTPDase activity measured. New Zealand white rabbits (N = 28) underwent bilateral iliofemoral artery balloon injury, followed by incubation with Ad-CD39, Ad-LacZ, or vehicle. Explanted vessels were analyzed for NTPDase activity and localization of CD39 expression by immunohistochemistry. Deposition of fluorescent-labeled platelets was studied 3 days after injury and vector treatment. RESULTS: In vitro, Ad-CD39 infection resulted in a greater than 40-fold increase in adenosine diphosphatase activity in ECs and a 3-fold increase in SMCs. In vivo, CD39 transgene expression localized to the luminal aspect of Ad-CD39--treated vessels. BI resulted in an acute reduction in vessel wall NTPDase activity (P <.05). Ad-CD39 augmented NTPDase activity when compared with vehicle or Ad-LacZ (P <.05). Platelet deposition on the injured arterial surface was modest and not different between Ad-CD39-- and Ad-LacZ--treated vessels. CONCLUSIONS: BI decreases native NTPDase activity, which can be augmented by adenovirus-mediated gene transfer of CD39. Further studies are required to determine whether targeted delivery of CD39 could convey thromboprotective properties to an injured vessel.

Acid Anhydride Hydrolases↗

PubMed2000

Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver.

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previously described in mammalian tissues. We report here the biochemical characterization of NTPDases in the pig liver. Optimum pH of catalysis is more acidic for this enzyme than for NTPDases (neutral or alkaline pH) found in other mammalian tissues. It is less sensitive to bile salts than the bovine spleen NTPDase. Calculated Km values for ATP and ADP (31 and 21 microM, respectively) are slightly higher than those reported for the latter enzyme. Electrophoretograms of these enzymes also show different migration patterns. Western blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimated molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A second antibody, generated against a different sequence of NTPDase I, does not recognize the liver enzyme, thereby indicating some differences in primary structure. Immunolocalization produced a strong signal on hepatocytes, epithelial cells of the bile duct system, and vascular cells. Immunoreactivity was variable among hepatocytes of different lobules and among hepatocytes within a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein.

Acid Anhydride Hydrolases↗

PubMed2000

Identification and characterization of a novel hepatic canalicular ATP diphosphohydrolase.

We have identified and characterized a novel ATP diphosphohydrolase (ATPDase) with features of E-type ATPases from porcine liver. Immunoblotting with a specific monoclonal antibody to this ectoenzyme revealed high expression in liver with lesser amounts in kidney and duodenum. This ATPDase was localized by immunohistochemistry to the bile canalicular domain of hepatocytes and to the luminal side of the renal ductular epithelium. In contrast, ATPDase/cd39 was detected in vascular endothelium and smooth muscle in these organs. We purified the putative ATPDase from liver by immunoaffinity techniques and obtained a heavily glycosylated protein with a molecular mass estimated at 75 kDa. This enzyme hydrolyzed all tri- and diphosphonucleosides but not AMP or diadenosine polyphosphates. There was an absolute requirement for divalent cations (Ca(2+) > Mg(2+)). Biochemical activity was unaffected by sodium azide or other inhibitors of ATPases. Kinetic parameters derived from purified preparations of hepatic ATPDase indicated V(max) of 8.5 units/mg of protein with apparent K(m) of 100 microM for both ATP or ADP as substrates. NH(2)-terminal amino acid sequencing revealed near 50% identity with rat liver lysosomal (Ca(2+)-Mg(2+))-ATPase. The different biochemical properties and localization of the hepatic ATPDase suggest pathophysiological functions that are distinct from the vascular ATPDase/cd39.

Adenosine↗

PubMed2000

Palmitoylation targets CD39/endothelial ATP diphosphohydrolase to caveolae.

Ectonucleotidases influence purinergic receptor function by the hydrolysis of extracellular nucleotides. CD39 is an integral membrane protein that is a prototype member of the nucleoside 5'-triphosphate diphosphohydrolase family. The native CD39 protein has two intracytoplasmic and two transmembrane domains. There is a large extracellular domain that undergoes extensive glycosylation and can be post-translationally modified by limited proteolysis. We have identified a potential thioester linkage site for S-acylation within the N-terminal region of CD39 and demonstrate that this region undergoes palmitoylation in a constitutive manner. The covalent lipid modification of this region of the protein appears to be important both in plasma membrane association and in targeting CD39 to caveolae. These specialized plasmalemmal domains are enriched in G protein-coupled receptors and appear to integrate cellular activation events. We suggest that palmitoylation could modulate the function of CD39 in regulating cellular signal transduction pathways.

Adenosine Triphosphatases↗

PubMed2000

Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1.

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.

Adenosine Triphosphatases↗

PubMed2000

Purification, characterization, and localization of an ATP diphosphohydrolase in porcine kidney.

Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.

Amino Acid Sequence↗

PubMed2000

Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain.

Extracellular nucleotides are ubiquitous extracellular mediators that interact with and activate nucleotide type 2 (P2) receptors. These receptors initiate a wide variety of signalling pathways that appear important for functional associations between neurons and glial cells and for the regulation of blood flow, haemostatic and inflammatory reactions in the brain. Ectonucleotidases are extracellular nucleotide-metabolizing enzymes that modulate P2 receptor-mediated signalling by the regulated hydrolysis of these agonists. A considerable number of ectoenzyme species with partially overlapping substrate and tissue distributions have been described. Major candidates for expression in the brain are members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase or CD39) family. The production of cd39-/- mice and specific reagents have enabled us to analyse the specific cellular distribution of NTPDase1 (CD39), the prototype member of the enzyme family, in the mouse brain. Using monospecific antibodies and enzyme histochemical staining, we have identified NTPDase1 as a major ectonucleotidase associated with both microglia and the endothelial and smooth muscle cells of the vasculature. NTPDase1 is not expressed by neurons and astrocytes. Additional unidentified ectonucleotidase functional activity is observed at lower levels throughout the brain parenchyma. NTPDase1 may regulate P2 receptor-mediated functions of microglia as well as influence nucleotide signalling between neurons or astrocytes that are associated with multiple microglial ramifications. The expression of NTPDase1 by cerebrovascular endothelial and smooth muscle cells also suggests involvement in the regulation of blood flow and thrombogenesis.

Acid Anhydride Hydrolases↗

PubMed1999

Identification and immunolocalization of two isoforms of ATP-diphosphohydrolase (ATPDase) in the pig immune system.

The occurrence of a variety of purine receptors in the immune system indicates that extracellular purines play important functional roles. Extracellular purine concentrations are, in great part, determined by ectonucleotidases, namely, the ATP diphosphohydrolase, also identified as CD39, a lymphocyte cell surface marker. The latter enzyme converts triphospho- and diphosphonucleosides to nucleoside monophosphates. In this study, high levels of ATPase and ADPase activities have been found in homogenates of the different pig lymphoid organs. Specific activities decreased in the following order: spleen > bone marrow > thymus > lymph glands. The parallel decrease in ATPase and ADPase activities, in the presence of sodium azide, indicated that an ATP diphosphohydrolase (ATPDase) was responsible for these activities. Particulate fractions, prepared from the different lymphoid organs by ultracentrifugation on a sucrose cushion, showed about a 10-fold enrichment of ATPDase activity. Identity of ATPDase was confirmed by electrophoretograms of the particulate fractions and Western immunoblots, with an antibody that recognizes ATPDases from different sources. Two isoforms of ATPDase were found (I and II), corresponding to molecular masses of 78,000 and 54,000, respectively, as estimated by SDS-PAGE. Immunohistochemical localization was carried out on these different organs: In spleen, reaction was found in both white and red pulps. A particularly intense reaction was put in evidence in nervous fibers of this organ. Immunolocalization also showed positive reactions with tonsilar lymphoid structures, diffuse lymphoid tissues, and nodules associated with stomach, duodenum, jejunum, and ileum. In addition, our observations establish the presence of ATPDase in lymphocytes and macrophages of the pig immune system.

Adenosine Triphosphatases↗

PubMed1999

Suppression of ATP diphosphohydrolase/CD39 in human vascular endothelial cells.

Vascular ATP diphosphohydrolase/CD39 is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities. Suppression of constitutive CD39 expression may result in elevated concentrations of ATP and ADP at the vascular interface that could predispose to thrombosis and inflammation. To study the effects of suppression of CD39 synthesis, stable 25-base antisense chimeric oligonucleotides targeting sequences at the 5' region of CD39 were designed. Transfection of these stable oligomers into cultured human endothelial cells resulted in dramatic decreases in levels of CD39 mRNA transcripts. Following transfection with antisense oligonucleotides, total ADPase activity fell from 26.0 +/- 3.1 in control cultures to 9.5 +/- 3.4 nmol of P(i) min(-1) (mg of protein)(-1) (p < 0.005); suppression of CD39 protein expression was also observed by Western blotting. Decreases in ATP diphosphohydrolase activity were associated with increases in concentrations of extracellular purine nucleotides released following stimulation of endothelial cells. Rates of initial hydrolysis of extracellular ATP released from purinergic agonist-stimulated endothelial cells decreased from 17.9 +/- 5.0 to 4.8 +/- 0.5 pmol min(-1) per 10(6) cells (p < 0.005) in antisense transfected cells. Therefore, CD39 regulates extracellular ATP concentrations and may be an important modulator of purinergic receptor activity in vascular endothelial cells.

Adenosine Diphosphate↗

PubMed1999

Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity.

CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.

Adenosine Triphosphatases↗

PubMed1999

Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation.

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro. This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39-deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

Adenosine Triphosphatases↗

PubMed1999

Analysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothelial cells, platelets and leukocytes.

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.

Adenosine Diphosphate↗

PubMed1999

Modulation of nucleoside [correction of nucleotide] triphosphate diphosphohydrolase-1 (NTPDase-1)cd39 in xenograft rejection.

BACKGROUND: There is increasing evidence showing that extracellular nucleosides [corrected] may be important mediators of vascular inflammation. Nucleoside [corrected] triphosphate diphosphohydrolase-1 (NTPDase-1, identical to CD39), the major vascular endothelial ectonucleotidase, is responsible for the hydrolysis of both extracellular ATP and ADP in the blood plasma to AMP. Studies were therefore conducted to evaluate the role of vascular NTPDase-1/cd39 in modulating platelet activation and vascular injury in cardiac xenografts. MATERIALS AND METHODS: Cardiac xenografts from both wild-type and cd39 knockout mice (C57BL/6 x 129 Svj) were transplanted into Lewis rats. Alterations in cd39 mRNA transcripts and NTPDase activity expression were evaluated in wild-type grafts in untreated rats and then following complement depletion and immunosuppression. Rejection responses were studied with both mutant and wild-type grafts in the following models: presensitization with or without complement depletion, complement depletion alone, and with chronic immunosuppression to induce long-term graft survival. RESULTS: NTPDase biochemical activity in wild-type xenografts rapidly decreased after transplantation but soon rebounded with graft survival. Elevated levels of cd39 mRNA with associated increases in NTPDase activity were observed in all long-term surviving wild-type grafts. Hyperacute xenograft rejection times were comparable in wild-type and mutant grafts but cd39-deficient grafts were subject to more rapid rejection and exhibited pronounced vascular injury in complement-depleted, presensitized rats. The cd39-deficient grafts in immunosuppressed recipients were subject to increased intravascular platelet sequestration and fibrin deposition; this resulted in focal myocardial infarction in long-term surviving mutant xenografts. CONCLUSIONS: Augmentation of NTPDase-1 activity may be an important adaptive response for graft survival. Our results suggest that NTPDase-1/cd39 influences pathways of vascular injury in cardiac xenografts.

Adenosine Triphosphatases↗