Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/ phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC 50 ∼ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.A utotaxin (ATX or NPP2) is a secreted nucleotide pyrophosphatase/phosphodiesterase (NPP) originally isolated as an autocrine motility factor from melanoma cells (1). ATX, a ∼120 kDa glycoprotein, is unique amongst the NPPs in that it functions as a lysophospholipase D (lysoPLD) that converts extracellular lysophosphatidylcholine (LPC) into the lipid mediator lysophosphatidic acid (LPA; mono-acyl-sn-glycero-3-phosphate) (2-5). LPA acts on specific G protein-coupled receptors and thereby stimulates the migration, proliferation, and survival of many cell types (6 and 7) (Fig. 1). ATX is produced by various tissues and is the major LPA-producing enzyme in the circulation. Newly produced LPA is subject to degradation by membranebound lipid phosphate phosphatases (LPPs) (8 and 9). However, little is known about the dynamic regulation of steady-state LPA levels in vivo.ATX is essential for vascular development (10 and 11) and is found overexpressed in various human cancers (12). Forced overexpression of ATX or individual LPA receptors promotes tumor progression in mouse models (13-16), while LPA receptor deficiency protects from colon carcinogenesis (17). In addition to its role in cancer, ATX-LPA signaling has been implicated in lymphocyte homing and (chronic) inflammation (18), fibrotic diseases (19 and 20), and thrombosis (21). Therefore, the ATX-LPA axis qualifies as an attractive target for therapies.Potent and selective ATX inhibitors are now needed as a starting point for the development of targeted anti-ATX/LPA therapy. Direct targeting of LPA receptors seems to be a less attractive strategy, since LPA acts on multiple receptors that show overlapping activities (2 and 6). Since the initial finding that ATX is subject to product inhibition by LPA and sphingosine 1-phosphate (S1P) (22), various synthetic phospho-and phosphonate lipids have been explored as ATX inhibitors (23-26). However, s...

The lipid mediator lysophosphatidic acid (LPA) is a potent regulator of vascular cell function in vitro, but its physiologic role in the cardiovasculature is largely unexplored. To address the role of LPA in regulating platelet function and thrombosis, we investigated the effects of LPA on isolated murine platelets. Although LPA activates platelets from the majority of human donors, we found that treatment of isolated murine platelets with physiologic concentrations of LPA attenuated agonist-induced aggregation. Transgenic overexpression of autotaxin/lysophospholipase D (Enpp2), the enzyme necessary for production of the bulk of biologically active LPA in plasma, elevated circulating LPA levels and induced a bleeding diathesis and attenuation of thrombosis in mice. Intravascular administration of exogenous LPA recapitulated the prolonged bleeding time observed in Enpp2-Tg mice. Enpp2 ؉/؊ mice, which have ϳ50% normal plasma LPA levels, were more prone to thrombosis. Plasma autotaxin associated with platelets during aggregation and concentrated in arterial thrombus, and activated but not resting platelets bound recombinant autotaxin/ lysoPLD in an integrin-dependent manner. These results identify a novel pathway in which LPA production by autotaxin/lysoPLD regulates murine hemostasis and thrombosis and suggest that binding of autotaxin/lysoPLD to activated platelets may provide a mechanism to localize LPA production.

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Phenotypic modulation of vascular smooth muscle cells (SMC) is essential for the development of intimal hyperplasia. Lysophosphatidic acid (LPA) is a serum component that can promote phenotypic modulation of cultured SMC, but an endogenous role for this bioactive lipid as a regulator of SMC function in vivo has not been established. Ligation injury of the carotid artery in mice increased levels in the vessel of both autotaxin, the lysophospholipaseD enzyme responsible for generation of extracellular LPA, and two LPA responsive G-protein coupled receptors 1 (LPA1) and 2 (LPA2). LPA1-/-2-/- mice were partially protected from the development of injury-induced neointimal hyperplasia, whereas LPA1-/- mice developed larger neointimal lesions after injury. Growth in serum, LPA-induced ERK activation, and migration to LPA and serum were all attenuated in SMC isolated from LPA1-/-2-/- mice. In contrast, LPA1-/- SMCs exhibited enhanced migration resulting from an upregulation of LPA3. However, despite their involvement in intimal hyperplasia, neither LPA1 nor LPA2 were required for dedifferentiation of SMC following vascular injury or dedifferentiation of isolated SMC in response to LPA or serum in vitro. Similarly, neither LPA1 nor LPA2 were required for LPA to elicit a transient increase in blood pressure following intravenous administration of LPA to mice. These results identify a role for LPA and two defined LPA receptors in regulating SMC migratory responses in the context of vascular injury, but suggest that additional LPA receptor subtypes are required for other LPA-mediated effects in the vasculature.