Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Gabor J. Tigyi, M.D., Ph.D.

Committee

Sarka Beranova-Giorgianni, Ph.D. John Fain, Ph.D. Suzanne Jackowski, Ph.D. Edwards Park, Ph.D.

Abstract

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 sn2‑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 sn1 over the sn2 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.

DOI

10.21007/etd.cghs.2011.0031

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