Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.

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

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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.The vascular ATP or NTP diphosphohydrolase (ATPDase or NTPDase; EC 3.6.1.5), 1 now known to be CD39, is a plasma membrane-bound enzyme that plays the dominant role in the hydrolysis of extracellular tri-and/or diphosphate nucleotides in blood (1, 2). Our recent data from cd39-null mice indicate that this specific ectonucleotidase also plays a pivotal role in the regulation of an ADP-purinoreceptor P2Y1 function; absence of ATPDase activity results in desensitization of this G protein-coupled receptor with profound effects on hemostasis and thromboregulation (3).Established topological models of CD39 suggest the presence of two transmembrane domains at both termini of the molecule and an extracellular loop containing a central hydrophobic region (1,4,5). The transmembrane domains of ATPDases appear to influence the formation of detergent-sensitive multimers (6). Examination of CD39 amino acid sequences reveals a total of 11 cysteine (Cys) residues, with an unpaired Cys 13 contained within the intracellular N terminus of the protein (4). Analysis of the CD39 sequence, by a computer algorithm PROSITE, further indicates six putative N-glycosylation sites with several potential casein kinase, cAMP/cGMP-dependent protein kinase or protein kinase C phosphorylation sites (7, 8).We have described several post-translational modifications of CD39. There are differences in the extent of glycosylation of human CD39 in endothelial cells, platelets, and leukocytes (9). Effects of limited serine proteolysis of native CD39 on ATPDase activity have been documented (5), and there is also a propensity for CD39 to undergo autophosphorylation reactions (data not shown). In addition, we have shown that cellular interactions with free fatty acids modulate ATPDase enzymatic activity in vitro and have postulated that acylation could also influence CD39 structure (10). Here we study this possibility and specifically examine palmitoylation, a reversible and potentially regulated post-translational modificat...