Autotaxin/lysopholipase D and lysophosphatidic acid regulate murine hemostasis and thrombosis

Abstract

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 approximately 50% 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.

FIGURE 1.

LPA induces aggregation and actin reorganization of human but not murine platelets. Human (A) or mouse platelets (B) (100,000/μl) were stirred in an aggregation cuvette, and 1 μm 1-oleoyl-sn-glycero-3-phosphate (designated LPA), or 1-O-hexadecyl-sn-glycero-3-phosphate (designated alkyl-LPA 16:0) was added. Arrows point to the timing of the addition of agonist. C, human or mouse platelets (20,000/μl) were incubated with fibrinogen-coated slides in the absence or presence of 1 μm LPA or 1 μm ADP for 30 min. Platelets were fixed, permeabilized with Triton X-100, stained with TRITC-phalloidin, and imaged with a 100× objective. Results are representative of those observed with platelets from six human donors and 10 experiments involving mouse platelets.

FIGURE 2.

LPA attenuates agonist-induced activation of murine platelets. Washed mouse platelets were treated with vehicle then agonist (A), 1 μm LPA then agonist (B), differing concentrations of thrombin (C), and 1 μm LPA then differing concentrations of thrombin (D), and aggregation was monitored by light transmission. Results are representative of those obtained with at least 10 separate preparations of platelets.

FIGURE 3.

LPA inhibition of murine platelet activation is dose- and cAMP-dependent. A, mouse platelets were incubated with the indicated dose of LPA, and aggregation was initiated by 0.05 units/ml thrombin. B, fibrinogen binding to platelets in whole blood was performed as described (30) in the presence of the indicated agonists. Results are graphed as the ratio of the mean fluorescent intensity in the presence and absence of LPA (mean ± S.D.; n = 6; ^*, p = 0.06; ^**, p = 0.011). C, platelet cAMP levels in murine platelets (1 × 10⁸) exposed to LPA. D, murine platelets were preincubated with the adenylyl cyclase inhibitor SQ22536 (SQ), which restored the ability of the PAR-4-activating peptide (PAR4;50 μm) to induce platelet aggregation in the presence of 1 μm LPA.

FIGURE 4.

Elevated autotaxin/lysoPLD activity in Ennp2-Tg mice. A, immunoblot analysis of autotaxin (ATX) in plasma from littermate or sex- and age-matched WT, Enpp2^+/-, and Empp2-Tg mice. The immunoblot is representative of results obtained in five separate experiments, and the mean ± S.D. from all the experiments is graphed. B, LysoPLD activity determined in plasma from WT (n = 3) and Enpp2-Tg (n = 3) mice by measuring generation of LPA from radiolabeled LPA. Results are presented as the mean ± S.D. Conditioned media from HEK cells that had not (control) or had been transfected with Enpp2 cDNA were used as negative and positive controls. C, LPA levels as measured by HPLC-tandem mass spectrometry in plasma from Enpp2-Tg mice were significantly elevated above those in WT plasma. Results are presented as mean ± S.D. (n = 3).

FIGURE 5.

Ennp2-Tg mice have a bleeding diathesis and impaired platelet-dependent thrombus formation after FeCl₃ injury of the carotid artery. A, tail bleeding time was determined in WT (n = 6) and Enpp2-Tg mice (n = 10). In all Ennp2-Tg mice, the tail had to be cauterized at 10 min to stop the bleeding (p < 0.001). B, blood flow was monitored in carotid arteries of WT (n = 6) and Ennp2-Tg (n = 6) mice after the application of FeCl₃, which elicited thrombotic occlusion as documented by a cessation of blood flow in 9 ± 2 min in WT mice. Up to 30 min after the addition of ferric chloride, no occlusion occurred in the Ennp2-Tg mouse (p < 0.001). At 30 min, vessel patency in the Ennp2-Tg mice was confirmed by occluding the vessel with a vascular clamp and recording the resultant drop in flow. C, Microscopic examination of carotid arteries 8 min after application of FeCl₃ revealed occlusive thrombus in WT vessels and mural thrombus in Enpp2-Tg vessels.

FIGURE 6.

Exogenous administration of LPA effects hemostasis in mice. A, the time to cessation of blood flow was determined in a tail bleeding assay using wild-type mice treated with either vehicle (n = 3) or LPA (20 nmol, n = 5, p < 0.05; 40 nmol n = 7; p < 0.05 versus control by one-way analysis of variance on ranks) by intraperitoneal administration 10-15 min before assessing bleeding time. B, the amount of blood loss from the tail of mice injected with different amounts of LPA was determined by monitoring the absorbance of hemoglobin in the solution (n = 8). C, thrombus formation in WT (n = 9) or Enpp2^+/- (n = 9) mice after application of 2.5% FeCl₃ to the carotid artery (p = 0.05 by Wilcoxon rank sum test).

FIGURE 7.

Autotaxin/lysoPLD accumulates in thrombus and incorporates into platelet aggregates. A, carotid arteries were collected from mice (n = 3) without or after application of 20% ferric chloride at the indicated times. Equal amounts of protein were separated by SDS-PAGE. Autotaxin (ATX)/lysoPLD and platelet integrin β3 associated with thrombus were detected by immunoblot analysis. Results are representative of those obtained in three separate experiments. B, carotid arteries were collected from WT (n = 3) and Enpp2-Tg mice (n = 3) after ferric chloride-induced thrombosis and probed for autotaxin as described above. The levels of autotaxin are graphed as -fold increase above WT (mean ± S.E.). C, platelets in plasma were stirred in the presence of ADP (1 μm), and aliquots were removed at the following times: unstim, before addition of ADP, 1′, 1 min after the addition of ADP; 5′, 5 min after the addition of ADP at the maximal aggregation; 10′, 10 min after the addition of ADP at a time when disaggregation has occurred. Platelets were separated from plasma by centrifugation and washed once, and autotaxin/lysoPLD associated with the platelets was detected by immunoblot analysis. The mean ± S.E. of four separate experiments are graphed.

FIGURE 8.

Autotaxin/lysoPLD binds activated platelets in a β3-integrin-dependent manner. A, recombinant human autotaxin (ATX)/lysoPLD was generated as previously described (41) and incubated at the indicated concentrations with microtiter plates. Gel-filtered human platelets without stimulation or with 10 μm ADP were incubated for 60 min at 37 °C in the presence of 2 mm CaCl₂ and 1 mm MgCl₂, and platelet adhesion was monitored as previously described (29, 48). Platelet adhesion to wells coated with fibrinogen (10 μg/ml) was performed as a positive control. Results are graphed as the mean ± S.D. from triplicates and are representative of results observed with 5 different donors. B, the anti-β3 antibody (Ab) 7E3 was included at 20 μg/ml in the adhesion assay, and platelets were stimulated with thrombin receptor activating peptide (TRAP, 15 μm). Results are graphed as the mean ± S.D. from triplicates and are representative of results observed with three different donors. C, immunoblot analysis of murine platelets isolated from WT (β3^+/+) or β3-deficient (β3^-/-) mice.