CD39/NTPDase1 Variants Identified in Human Neutrophils Regulate Antithrombotic Activity.

Abstract

Abstract Blood-borne cellular elements expressing ectonucleotidase activity have been shown to regulate platelet activation and recruitment in response to agonists. In particular, exposure of a platelet releasate to isolated neutrophils (PMN) results in loss of its platelet activating activity in a subsequent assay (Valles et al, J Clin Invest1993, 92:1357–1365). Whereas expression of CD39 on vascular endothelial cells has been well characterized, expression on leukocytes has been less well studied. Freshly prepared lymphocyte and PMN cell populations were evaluated for both cell surface expression of CD39 and ectonucleotidase activity. FACS analysis showed that 98% of PMN were positive for CD39 compared to only 20% of lymphocytes. In addition, neutrophils stained more intensely, indicating the presence of a higher quantity of cell surface-expressed CD39. Interestingly, neutrophils exhibited only 1/3 of the ATPase and 1/2 of the ADPase activities of the same number of lymphocytes, although the latter are thought to have greater antithrombotic capacity. RT-PCR products from total RNA isolated from lymphocytes and PMN were sequenced. This revealed alternately spliced CD39 mRNA species present in PMN at levels equal to that of CD39 mRNA. In contrast, lymphocytes, which showed much higher levels of CD39 mRNA, expressed these variants at much lower levels. RACE analyses of cDNAs generated from total RNA demonstrated two CD39 gene-derived mRNAs. Each was comprised of an alternate 3′ segment lacking the C-terminal transmembrane domain, and distinguished by an internal deletion. Myc- and Flag-tagged constructs expressed in COS cells resulted in cell surface expression of the respectively tagged variants (immunocytochemistry, western blot analyses of plasma membrane preparations). Membrane preparations assayed for enzyme activity revealed no apyrase activity for either molecule expressed alone or together. Co-transfection of CD39 with equal amounts of either construct singly or in combination resulted in a 30-50% decrease in ATPase activity compared to CD39 alone. Similarly, CD39 co-expressed with either construct alone lost 75–90% of its ADPase activity. Unexpectedly, co-transfection of CD39 with both variants together resulted in a 20–40% increase in ADPase activity. Glutaraldehyde cross-linking of membrane preparations from triply transfected COS cells followed by immunoprecipitation and western blot analyses demonstrated the presence of all three species in higher order complexes. Thus, both variants can simultaneously associate with CD39, generating hetero-multimers with altered substrate preference and catalytic efficiency compared to CD39 tetramers. These observations add to our understanding of the regulation of ectonucleotidase activity at the cell surface. The balanced expression of CD39 and its two identified variants may underlie the anti-platelet activity of neutrophils previously reported. The finding that association of CD39 with either construct alone results in near complete loss of ADPase activity with only partial diminution of ATPase activity suggests a possible etiology for a pro-thrombotic phenotype.