Validation of an autotaxin enzyme immunoassay in human serum samples and its application to hypoalbuminemia differentiation

Nakamura, Kazuhiro; Igarashi, Koji; Ide, Kazufumi; Ohkawa, Ryunosuke; Okubo, Shigeo; Yokota, Hiromitsu; Masuda, Akiko; Oshima, Noriko; Takeuchi, Takumi; Nangaku, Masaomi; Okudaira, Shinichi; Arai, Hiroyuki; Ikeda, Hitoshi; Aoki, Junken; Yatomi, Yutaka

doi:10.1016/j.cca.2007.10.005

Cited by 104 publications

(130 citation statements)

References 43 publications

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“…The increase in LPA production was dependent on the concentration of added ATX with a half-maximal effect observed at concentrations of ϳ100 nM (Fig. 1B), which is in the range of the concentration of ATX reported in human plasma (29). When incubated with a maximally effective concentration of ATX, LPA generation by platelets was sustained at times up to 20 min (Fig.…”

Section: Synergistic Effect Of Platelet Activation and Atx On Lpamentioning

confidence: 70%

Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells

Fulkerson

Sunkara

et al. 2011

Journal of Biological Chemistry

138

136

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Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA).We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin ␣IIb␤ 3 . The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet ␤1 and ␤3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ϳ10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A 2 , and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin ␤ 3 . The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors.

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Section: Synergistic Effect Of Platelet Activation and Atx On Lpamentioning

confidence: 70%

Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells

Fulkerson

Sunkara

et al. 2011

Journal of Biological Chemistry

138

136

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“…14 A collaborative group of researchers partially purified membrane fractions of CHO cells stably transfected with NPP1, NPP2, or NPP3 to probe their activity with Ap3A, and all were observed to hydrolyze Ap3A with K m s in the low M range. Our studies investigated NPP family members suggested by the literature to be present in vascular cells 17,29 -NPP2 and NPP4-and used a baculovirus expression system to produce soluble, easily purified proteins. In contrast to previous reports, we have seen no evidence that NPP2 is capable of hydrolyzing Ap3A either by steady state enzymatic studies (data not shown) or biologic assays with PRP ( Figure 5C).…”

Section: Discussionmentioning

confidence: 99%

NPP4 is a procoagulant enzyme on the surface of vascular endothelium

Albright

Chang

Robert

et al. 2012

Blood

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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 se- IntroductionThe formation of a platelet-rich thrombus stabilized by fibrin crosslinking is the final common pathway for arterial thrombosis, the most common disease process affecting adults in the United States. The first step in thrombus formation consists of platelet adhesion to the exposed subendothelial extracellular matrix at the site of vascular injury. Here, circulating blood platelets bind collagen via their GPVI receptors, and bind von Willebrand factor via GPIb, triggering inside-out activation of other surface integrins and the release of platelet granule contents into the extracellular space. 1 The release of preformed molecules stored in platelet-dense granules such as ADP, serotonin, and ionized calcium, then amplifies the clotting reaction beyond the platelet monolayer bound on the collagen surface to circulating platelets in the immediate vicinity of the damaged endothelium. This amplification is further augmented by the secretion of thromboxane A2. ADP binding to purinergic receptors on the platelet surface (P2Y1 and P2Y12) induces rapid calcium influx and mobilization resulting in platelet shape change, activation, and partial degranulation thus promoting platelet aggregation. On granule release, local ADP concentrations are estimated to exceed 500M, but ADP is quickly metabolized by ectoenzymes on the surface of endothelial cells (such as CD39 2,3 ) and soluble phosphohydrolyases in blood plasma which attenuate the prothrombotic response. [4][5][6][7] To sustain the clotting reaction, a slow and steady source of ADP at the site of the growing thrombus is required. Another preformed chemical released by platelet-dense granules at high concentrations, diadenosine triphosphate (Ap3A), has an ill-defined role in thrombus formation but has been suggested to provide a source of long-lasting ADP at the site of vascular injury.Ap3A is stored within platelet granules at high concentrations (ϳ20-30mM) and is released with ADP and ATP into the blood during thrombin-induced platelet aggregation at concentrations thought to range between 40 and 100M 8 (see supplemental Table ...

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“…n = 5 experiments in each group. *P < 0.05. serum ENPP2 levels in large numbers of human samples (15). The results showed that serum ENPP2 levels are reduced in obese subjects, and multivariate analysis indicated ENPP2 to be an independent parameter associated with BMI (Supplementary Tables 1 and 2).…”

Section: Enpp2 Expression In Subcutaneous Fat and Serum Levels Are Rementioning

confidence: 96%

ENPP2 Contributes to Adipose Tissue Expansion and Insulin Resistance in Diet-Induced Obesity

et al. 2014

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Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2 +/2 mice and adipocyte-specific Enpp2 knockout mice fed a highfat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease.Until recently, adipose tissue was viewed as a passive energy storage organ, but with the discovery of leptin and the adipose-derived humoral factors now known as "adipokines," it has become apparent that adipose tissue is an active endocrine organ that is essential for energy homeostasis (1). Moreover, obese adipose tissue secretes various inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), whose activities are known to contribute to the development of metabolic and cardiovascular diseases (2).Enpp2, also designated autotaxin, phosphodiesterase I a/autotaxin, and nucleotide pyrophosphatase/phosphodiesterase 2, was originally discovered as an autocrine motility-stimulating factor released from cancer cells (3). ENPP2 catalyzes the conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA), which exerts a variety of biological effects, in part via G-protein-coupled receptors (3,4). In addition, the COOH-terminal noncatalytic domain of ENPP2 also has biological effects independent of LPA (3). Homozygous Enpp2-deficient mice die in utero due to profound vascular defects, but heterozygous Enpp2-deficient (Enpp2 +/2 ) mice are apparently healthy, with plasma LPA levels about half those in wild-type (WT) mice (5). Enpp2 is reportedly expressed in mouse adipose tissue and 3T3-F442A preadipocytes, and medium conditioned by Enpp2-expressing COS7 cells increased proliferation of 3T3-F442A cells. Recently, Dusaulcy et al. (6) reported that adipocytespecific Enpp2 knockout (KO) mice fed a high-fat diet showed greater adiposity and less systemic insulin resistance

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Validation of an autotaxin enzyme immunoassay in human serum samples and its application to hypoalbuminemia differentiation

Cited by 104 publications

References 43 publications

Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells

Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells

NPP4 is a procoagulant enzyme on the surface of vascular endothelium

ENPP2 Contributes to Adipose Tissue Expansion and Insulin Resistance in Diet-Induced Obesity

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