title: NPP4 is a procoagulant enzyme on the surface of vascular endothelium.
abstract: Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Albright
authors.forename: William C
authors.initials: WC
authors.lastname: Chang
authors.forename: Donna
authors.initials: D
authors.lastname: Robert
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Ornstein
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Cao
authors.forename: Lynn
authors.initials: L
authors.lastname: Liu
authors.forename: Meredith E
authors.initials: ME
authors.lastname: Redick
authors.forename: J Isaac
authors.initials: JI
authors.lastname: Young
authors.forename: Enrique M
authors.initials: EM
authors.lastname: De La Cruz
authors.forename: Demetrios T
authors.initials: DT
authors.lastname: Braddock
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000328:Adult; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D003029:Coagulants / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000293:injuries / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007313:Insecta; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D011755:Pyrophosphatases / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D014018:Tissue Distribution
chemical_list: D003029:Coagulants; D015226:Dinucleoside Phosphates; C020733:diadenosine tetraphosphate; C011440:diadenosine triphosphate; D000244:Adenosine Diphosphate; C577867:ENPP4 protein, human; D010727:Phosphoric Diester Hydrolases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase