Localizing LPA Production

Science Signaling  11 Oct 2011:
Vol. 4, Issue 194, pp. ec285
DOI: 10.1126/scisignal.4194ec285

Production of the bioactive lipid lysophosphatidic acid (LPA), which regulates endothelial and vascular smooth muscle cell function and contributes to platelet activation, is mediated by the secreted enzyme autotaxin (ATX) through the hydrolysis of lysophosphatidylcholine (LPC). LPA production is greater in platelet-rich plasma relative to platelet-poor plasma, and thrombocytopenia caused by an antibody that recognizes platelets or by a small-molecule inhibitor of the platelet fibrinogen receptor integrin αIIbβ3 is associated with reduced circulating LPA. Fulkerson et al. found that the production of LPA by platelets stimulated with thrombin was blocked by inhibition of ATX activity and was enhanced by the addition of recombinant ATX. Furthermore, using a catalytically inactive form of ATX (ATX-T210A) to avoid complications due to stimulation of the platelets by the produced LPA, binding of platelets to ATX-coated dishes was inhibited by the absence of conditions that blocked activation of β1- or β3-containing integrins. Forced expression of β3 integrin in Chinese hamster ovary (CHO) cells in the presence of an integrin activator (Mn2+) promoted binding of these cells to ATX. Saturable binding of radiolabeled ATX was also detected on platelets in suspension, and ATX binding was reduced by the addition of fibrinogen or echistatin, a peptide that competitively inhibits ligand binding to integrins. ATX or the catalytically inactive mutant reduced platelet adhesion in response to an agonist, whereas platelet adhesion was enhanced by the addition of fibrinogen. Catalytically inactive ATX prevented this fibrinogen-mediated enhancement, consistent with competition of these two molecules for the same receptor. At low concentrations of fibrinogen, wild-type ATX promoted adhesion and spreading of platelets. Mutational analysis identified the SMB (somatomedin B-like) 2 domain, and binding of peptide fragments containing only the two SMB domains to CHO cells expressing β3 integrin showed that these domains were necessary and sufficient for binding. Finally, integrin activation of the integrin-expressing CHO cells promoted ATX activity, and this was further enhanced if the cells were pretreated with phospholipase A2 to increase the production of the ATX substrate LPC. The enhanced production of LPA was not detected in the presence of integrin-blocking antibodies or the use of mutant forms of ATX that could not bind integrin. Thus, the authors propose that by binding to activated integrins, ATX is recruited to platelets and cells, where it locally produces the signaling molecule LPA.

Z. Fulkerson, T. Wu, M. Sunkara, C. Vander Kooi, A. J. Morris, S. S. Smyth, Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells. J. Biol. Chem. 286, 34654–34663 (2011). [Abstract] [Full Text]