Platelet activation initiates an upsurge in 18:2 and 20:4 lysophosphatidic acid (LPA) production. The biochemical pathway responsible for LPA production during blood clotting is not fully understood. We have purified a phospholipase A₁ (PLA₁) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate‑biotin affinity labeling and proteomics. We identified acyl‑protein thioesterase 1 (aka. lysophospholipase A1, accession code O75608) as a novel PLA₁. Addition of this recombinant PLA₁ significantly increased the production of sn‑2‑esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We next examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acylmigration regioisomers of oleyl‑sn‑glycero‑3‑phosphocholine (LPAF) were synthesized. ATX preferred the sn‑1 over the sn‑2 regioisomer of LPAF. We propose the following LPA production pathway in blood: 1) Activated platelets secrete PLA₁. 2) PLA₁generates a pool of sn‑2 lysophospholipids. 3) These newly generated sn‑2 lysophospholipids undergo acyl migration to yield sn‑1 lysophospholipids, which are the preferred substrates of ATX. 4) ATX cleaves the sn‑1 lysophospholipids to generate sn‑1 LPA species predominant with 18:2 and 20:4 fatty acids. Advisors/Committee Members: Gabor J. Tigyi, M.D., Ph.D..

Lysophosphatidic acid (LPA) and its ether analog alkyl glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1 hour without injury into the carotid arteries of LPA1, LPA2, LPA1&2 double knockout, and Mx1Cre-inducible conditional PPARγ knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and "-smooth muscle actin markers. In LPA1, LPA2, LPA1&2 GPCR knockouts, Mx1Cre transgenic, PPARγ fl/- , and uninduced Mx1Cre#PPAR! fl/- mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARγ -/- knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon-injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARγ in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express !-smooth muscle actin. Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature’s simplest phospholipids, is found in cells from slime mold to humans and has largely unknown function. We find that CPA is generated in mammalian cells in a stimulus coupled-manner by phospholipase D2 (PLD2), and binds to and inhibits the nuclear hormone receptor PPAR! with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPAR! target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in vivo. Inhibition of PLD2 by shRNA, a dominant negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARγ inhibition. We conclude that CPA is a novel second messenger and a physiological inhibitor of PPARγ, revealing that PPARγ is regulated by endogenous agonists as well as by antagonists. PPAR" is a nuclear hormone receptor related to many human diseases, including obesity, atherosclerosis, diabetes, and cancers. Recent studies have provided evidence that LPA and its analog AGP activate PPARγ. On the… Advisors/Committee Members: Gabor J. Tigyi, M.D., Ph.D..