The Biochemical Pathway Leading to Lpa Generation Upon Blood Coagulation

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

Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARγ by cyclic phosphatidic acid

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature\u27s simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here 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 PPARγ with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARγ 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 a rat model 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 second messenger and a physiological inhibitor of PPARγ, revealing that PPARγ is regulated by endogenous agonists as well as by antagonists. © 2010 Elsevier Inc

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipaseD activity, which leads to generation of the growth-factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4-tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues block ATX activity with Ki values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed-mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers invitro in a dose-dependent manner. The average terminal half-life for compound 22 is 10±5.4h and it causes a long-lasting decrease in plasma LPA levels. Compounds 22 and 30b significantly decrease lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The 4-substituted benzylphosphonic acids and 6-substituted naphthalen-2-ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX-LPA-LPAR axis both invitro and invivo. Inhibiting the ATX-LPA-LPAR axis: New 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues were synthesized, and the most potent ATX inhibitors, 22 and 30b, show outstanding invivo profiles by diminishing lung metastases of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment model. These two lead compounds effectively inhibit the ATX-LPA-LPAR axis both invitro and invivo. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The phospholipase A 1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purifi cation of a phospholipase A 1 (PLA 1 ) from thrombin-activated human platelets using sequential chromatographic steps followed by fl uorophosphonate (FP)-biotin affi nity labeling and proteomics characterization that identifi ed acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA 1 . Addition of this recombinant PLA 1 signifi - cantly increased the production of sn -2-esterifi ed polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl- sn -glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn -1 to the sn -2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1 ) Activated platelets release PLA Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purifi cation of a phospholipase A 1 (PLA 1 ) from thrombin-activated human platelets using sequential chromatographic steps followed by fl uorophosphonate (FP)-biotin affi nity labeling and proteomics characterization that identifi ed acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA 1 . Addition of this recombinant PLA 1 signifi - cantly increased the production of sn -2-esterifi ed polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl- sn -glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn -1 to the sn -2 regioisomer of lyso- PAF. We propose the following LPA production pathway in blood: 1 ) Activated platelets release PLA 1 ; 2 ) PLA 1 generates 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; and 4 ) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.-Bolen, A. L., A. P. Naren, S. Yarlagadda, S. Beranova- Giorgianni, L. Chen, D. Norman, D. L. Baker, M. M. Rowland, M. D. Best, T. Sano, T. Tsukahara, K. Liliom, Y. Igarashi, and G. Tigyi. The phospholipase A 1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation. J. Lipid Res. 2011. 52: 958-970.1 ; 2 ) PLA 1 generates 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; and 4 ) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids. Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation[S]

Platelet activation initiates an upsurge in polyun­saturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A1 (PLA1) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; 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 examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA1; 2) PLA1 generates 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; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids