Overexpression of the Plasma Lysophospholipase D Autotaxin Reveals a Novel Pathway for Regulation of Hemostasis in Mice.

The cell-motility factor autotaxin (autotaxin/lysoPLD or Enpp2) is an enzyme with both lysophospholipase D (lysoPLD) and nucleotide pyrophosphatase/phosphodiesterase activities and has recently been identified as a critical component of the major pathway for generation of the bioactive lipid mediator lysophosphatidic acid (LPA) in the blood. Transgenic FVB mice overexpressing autotaxin/lysoPLD under the control of the alpha1-antitrypsin promoter (Enpp2-Tg mice) exhibited elevated plasma autotaxin/lysoPLD protein, lysoPLD activity and LPA levels, with no detectable alteration in plasma levels of ATP, ADP, and adenosine. Ennp2-Tg mice displayed a pronounced bleeding diathesis characterized by markedly prolonged tail bleeding time (>10 min versus 3.2 ± 2.7 min in wild-type mice; p <0.001) and variable protection from thrombosis. Ennp2-Tg mice did not form occlusive thrombus in the carotid artery within 30 min following application of ferric chloride (versus a time to thrombotic occlusion of 9 ± 2 min in wild-type mice; p<0.001), and histologically, only mural thrombus was present along the ferric chloride-treated carotid arteries of the Ennp2-Tg mice. However, intravascular thrombosis in response to intravenous injection of collagen + epinephrine was normal in Ennp2-Tg mice. Enpp2-Tg mice exhibited normal platelet counts (928 ± 184 versus 808 ± 156 x 10³/mm³), and normal platelet membrane protein expression, platelet aggregation, and coagulation parameters in ex vivo studies. Although LPA is a weak activator of platelets isolated from the majority of human donors, we found that treatment of isolated murine platelets with physiologically relevant levels of LPA (1μM) attenuated ADP (1μM)-induced and low-dose thrombin (0.01 – 0.05U/ml)-induced aggregation, but did not alter aggregation elicited by higher doses of thrombin or collagen. Treatment of murine platelets with LPA also elevated intraplatelet cAMP levels. Moreover, systemic administration of LPA to mice recapitulated the prolonged bleeding time observed in Enpp2-Tg mice. These results confirm the importance of autotaxin/lysoPLD as the main determinant of circulating LPA levels and suggest a novel pathway for regulation of murine hemostasis and thrombosis involving the autotaxin/lysoPLD-LPA axis.