ADP doc - Supplements - Haematologica

54Figure 1. UVEC metabolism of ADP. HUVEC were incubatedfor 5 min with 50 µM [ 14 C]-ADP. ADP metabolites were separatedby radio-TLC. The activity of 5’nucleotidase, as wellas adenosine deaminase is inferred from the rapid appearanceof adenosine and inosine in these and other scans.COS cells do not possess ecto-ATPase, -ADPase or5’nucleotidase (data not shown). Was performed with AnInstantImager ® (Packard Instrument Co., Meriden, CT, USA)were used for detection and quantification.plasma (PRP) to result in addition of 5 µM ADP if theADP had not been metabolized. However, the datademonstrated that 14 C-ADP and induction of plateletactivation decreased concurrently and rapidly. Inaddition, AMP, accumulated transiently, was furthermetabolized to adenosine and then deaminated toinosine (Figure 1). 1 We next sought to identify themolecule(s) responsible for this presumed ADPaseactivity. Initially, we established that the HUVECADPase was a membrane-associated ecto-nucleotidaseof the E-type. 4 Characteristics of this enzymeincluded Ca/Mg dependence, ineffectiveness of specificinhibitors of P-, F-, and V-type ATPases, and thecapacity to metabolize both ATP and ADP, but notAMP. Such properties identified the HUVEC enzymeas an apyrase (ATP diphosphohydrolase), ATPDase,EC3.6.1.5. 4 At the time, ecto-nucleotidase researchwas severely hindered by difficulties in protein isolation.This was due to the low abundance of the protein(s)in a setting of high enzyme activity and theirsensitivity to denaturing agents. 4-7 The HUVECenzyme is indeed an example of this.A new nomenclature has recently been proposed tounify usage in this rapidly evolving and broadeningfield. 8 According to this nomenclature, HUVEC ecto-ADPase/CD39 is human E-NTPDase-1.In 1996, a soluble apyrase was purified from potatotubers, and its cDNA cloned. 9 Sequence analysisrevealed 25% amino acid identity and 48% aminoacid homology with human CD39. 9 CD39 had beencloned as a cell-surface glycoprotein, 10 expressed onactivated B-cells, NK cells, and subsets of T-cells aswell as on some HUVEC cell lines. 11 Nucleotidaseswith homology to CD39 and potato apyrase areexpressed throughout nature in species as varied asthe garden pea, C. elegans and Toxoplasma. 9 Interestingly,at least 4 regions within these molecules hadextraordinary homology, and were designatedapyrase conserved regions (ACR). 9 From thesereports, we proposed that HUVEC ADPase is identicalto CD39. 12 This was based on the following observations:more than 95% of the ADPase activity froman ADPase preparation purified from HUVEC membranescan be immunoprecipitated with any of severalanti-human CD39 antibodies. Confocal microscopyand indirect immunofluorescence studies localizedCD39 to the HUVEC cell surface. Most importantly,when we transfected COS cells with a vectorcontaining the cDNA for either human or murineCD39, we could demonstrate expression of bothCD39 and ecto-ADPase activity on the COS cell surface.Polymerase chain reaction (PCR) analyses usingeither authentic human CD39 cDNA or cDNA synthesizedfrom HUVEC mRNA resulted in products ofidentical size for each of four different CD39-specificprimer pairs. Sequencing of the PCR products confirmedtheir identity in each instance. The PCR productsencompassed 75% of the coding region ofCD39, including the original 4 ACR (apyrase domain,Figure 2). In addition, Northern analyses demonstratedthat HUVEC and MP-1 cells (from whichCD39 was originally cloned) contained same sizedmessages for CD39. Protein purification studies ofecto-ATPDases from different cell sources werereported from other laboratories as well. 13,14 Of criticalimportance were experiments in which COS cellstransfected with human CD39 cDNA acquired theability to block ADP-induced platelet aggregation. 12This occurred with COS cells transfected with eitherhuman or murine CD39. Transfectants metabolizedADP to AMP within 3 minutes. These observationsare especially pertinent to the time frame of eventsleading to formation of a hemostatic platelet plug orthrombus. We know that platelet adhesion to injuredsubendothelium leads to immediate release of ADPand recruitment of additional platelets to form anocclusive thrombus in less than 4 minutes. Thischronology parallels the time-course we observed forplatelet inhibition by CD39-expressing cells and wasalso commensurate with their respective ADPaseactivities (Figure 3). These results amplify the importanceof CD39 as a thromboregulator. They also representthe first direct demonstration of a physiologicalfunction for CD39 as an ADPase, i.e. blockade ofplatelet responsiveness to the prothrombotic agonistADP via its metabolism to AMP. This phenomenonmight represent evolution of an endothelial mechanismtargeted toward metabolism of prothromboticplatelet-derived ADP in preference to ATP, therebycontrolling excessive platelet accumulation. The biologicalproperties of CD39 suggested a novel strategyfor therapeutic intervention. While aspirin treat-Haematologica vol. 85 (the Platelet ADP Receptors supplement), June 2000