Characterization of Nucleotide Pyrophosphatase/Phosphodiesterase 4, a Dinucleotide Hydrolase, and Its Promotion of Platelet Aggregation.

摘要

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are a family of enzymes found in eukaryotes and bacteria that hydrolyze nucleotide or lipid phosphodiesters. In eukaryotes, they are expressed as secreted or membrane-bound proteins and are known to regulate a diverse range of physiological processes, including cardiovascular function, tumor development, and biomineralization. Despite their predicted structural similarity, NPP substrate specificity varies greatly: NPP1 and NPP3 are nucleotide hydrolases, and NPP2, NPP6, and NPP7 are phospholipid hydrolases, while NPP4 and NPP5 are uncharacterized. Two structures of NPPs have been solved: one from the bacterium Xanthomonas axonopodis pv. citri, and the mouse and rat isoforms of NPP2.;We developed a baculovirus expression system in insect cells for expressing human NPPs as soluble secreted proteins. The extracellular portion of each gene is expressed with signal peptides, either native to the protein or cloned from NPP2, targeting the protein for secretion. A tobacco etch virus (TEV) protease-cleavable 9x-histidine tag was cloned onto the C-terminus of each gene, allowing the protein to be purified by two rounds of Ni-NTA affinity column purification. I successfully cloned and expressed NPP1, NPP2, NPP4, and NPP5, yielding several milligrams of highly pure protein per liter of cell culture.;With pure soluble recombinant protein, I could enzymatically characterize human NPPs. I developed a coupled assay using alkaline phosphatase and a malachite green detection system to identify nucleotide and phospholipid substrates for NPP2, NPP4, and NPP5. NPP2 hydrolyzes lysophospholipids while it, contrary to previous reports, does not hydrolyze dinucleotide polyphosphates. NPP4, in contrast, hydrolyzes dinucleoside polyphosphates and not phospholipids. NPP5 did not hydrolyze any nucleotide or phospholipid substrates tested. I thoroughly characterized the steady-state activity of NPP4 using three complementary assays: the coupled assay as described above, high performance liquid chromatography analysis of substrates and products, and, for Ap3A, Ap4A, and pNP-TMP, absorbance change monitoring. Under steady-state conditions, NPP4 hydrolyzes Ap3A with a KM of 843.13 +/- 131.9 muM and a kcat of 4.247 +/- 0.403 s-1 NPP4 -1, and hydrolyzes Ap4A with a KM of 209.90 +/- 2.81 and a kcat of 0.784 +/- 0.00736 s-1 NPP4-1. These are the first enzymatic characterizations of NPP4.;We have determined the structure of NPP4 using x-ray crystallography to 1.54 angstrom resolution bound with its enzymatic product AMP. This is the first structure of a human NPP to be solved. The overall architecture and the active site catalytic residues and metals are arranged similarly to NPP2 and Xac NPP. The active site binding pocket contains a loop absent in NPP2 forming a back wall, preventing longer substrates from binding. The binding pocket of NPP4 also contains tyr154, which forms a pi-pi stacking interaction the bound adenosine, stabilized by phe71. In other NPP family members such as NPP5, the homologous residues make the binding pocket smaller and electrostatically unfavorable for AMP.;The physiological role and substrate of NPP4 is unknown. We identified Ap3A as an NPP4 substrate. Ap3A is secreted from platelets, adrenal chromaffin cells, and brain synaptic terminals, so we investigated whether NPP4 is present in any of their microenvironments. Using immunofluorescence, we found NPP4 in the human brain vascular endothelium. Diadenosine polyphosphatase hydrolase activity has been reported in vascular endothelia, but a specific enzyme has never been identified. Diadenosine polyphosphates and their hydrolysis products ADP and ATP are agonists and antagonists of platelet aggregation respectively. As NPP4 is a diadenosine polyphosphate hydrolase found on vascular endothelia, we hypothesize that it is an in vivo regulator of platelet aggregation. Using light-transmission aggregometry, we found that NPP4 and Ap3A stimulate platelet aggregation in a dose-dependent manner, showing complete irreversible aggregation at 80 nM NPP4 and 80 muM Ap3A. Platelets can have dense granule secretion deficiencies, either congenital, such as storage pool disease, or acquired, such as through the administration of nonsteroidal anti-inflammatory drugs (NSAIDs), reducing platelets' ability to aggregate. In platelets with dense granule secretion deficiencies, 40-50 nM of exogenous NPP4 is able to normalize platelet aggregation.;Antiplatelet therapies, such as low-dose aspirin and clopidogrel, are used to prevent heart attacks, strokes, and other cardiovascular diseases, the top causes of death in the developed world. However, as current antiplatelet therapies inhibit platelet function systemically, they increase the risk by 1 percent of major bleeding requiring transfusion. Targeting NPP4 for inhibition may be a means to localize an antithrombotic effect to the site of thrombus formation. In contrast, exogenous NPP4 may be a means of treating bleeding disorders resulting from intrinsic platelet granule defects. Our studies of NPP4 give us a model and techniques for studying the roles and characteristics of human NPP-family enzymes.