Effect of aggregation on the regulation of cytosolic phospholipase A₂ activity in human platelets

Author

Xiao-Fen Wang

Date of Award

5-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Comparative and Experimental Medicine

Major Professor

Roger C. Carroll

Committee Members

Albert T. Ichiki, Philip N. Bochsler

Abstract

Ro 44-9883, a potent and selective GPIIb/IIIa antagonist, is an antithrombotic peptidomimetic developed using the arginine-glycine-aspartate-serine (RGBS) sequence as a chemical lead compound. Inhibition of aggregation, as detected by an aggregometer, by Ro 44-9883 resulted in complete inhibition of ¹⁴C-serotonin secretion. This correlated with an inhibition of thromboxane A₂(TxA₂) formation, detected by EIA, which was induced by weak agonists such as ADP or low dose thrombin receptor agonist peptide (TRAP). The inhibition of serotonin secretion as well as TxA₂ formation was overcome by higher doses of TRAP. Ro 44-9883 had no effect on serotonin secretion in Glanzmann's thrombasthenic platelets. Inhibition of serotonin secretion was not completely reversed by the TxA₂ mimetic, U46619, or exogenously added arachidonic acid, but additionally required lysophosphatidic acid. Interestingly, Ro 44-9883 inhibition of TxA₂ formation was not due to a lack of cytosolic phospholipase A₂ (cPLA₂) activation as assayed in vitro. Moreover, blocking aggregation with Ro 44-9883 had no effect on phospholipase C generated IP₃ as measured by ³H-IP₃ radioreceptor assay. spectrophotometrically measured cytosolic calcium flux and pH changes, diacylglycerol formation, as detected by ³²P-labeling of the phosphatidic acid pool and thin-layer chromatography (TLC), nor on protein kinase C and myosin kinase activities, as detected by ³²P-labeling, SDS-PAGE and autoradiography. However, tyrosine phosphorylation of pl25^FAK (focal adhesion kinase) in both low and high dose TRAP-stimulated platelets was blocked by Ro 44-9883 pretreatment, as observed by SDS-PAGE and western blotting. These results suggest that aggregation is specifically required for weak agonists induced intracellular activity of cPLA₂, possibly by regulating phospholipid substrate availability or interaction of CPLA₂ with the membrane. Our study also suggested unactivated platelets have a major portion of cPLA₂ associated with the membrane, which is not mediated by actin, and possibly mediated by interaction by an unknown mechanism with several membrane proteins, as evaluated by immunoprecipitation, SDS-PAGE and western blotting. Platelet activation results in cPLA₂ phosphorylation and a shift of cPLA₂ from the membrane to the cytosol with a large portion associated with the cytoskeleton since this CPLA₂ could be released by DNase I treatment to depolymerize actin filaments. This cytosolic/cytoskeletal fraction also contained most of the phosphorylated CPLA₂ with no ³²P-labeled CPLA₂ found in membrane fractions, as detected by ³²P-labeling, IPPT, SDS-PAGE, and autoradiography. Blocking aggregation neither affected phosphorylation of CPLA₂, nor the redistribution of CPLA₂ between the cytosol and the membrane. These results suggest that membrane protein clustering in connection with aggregation, perhaps related to tyrosine phosphorylation of pl25^FAK, is required for the phospholipid substrates of CPLA₂ to be exposed. Additionally, these results suggest a more complex regulation of CPLA₂ than previously proposed.

Recommended Citation

Wang, Xiao-Fen, "Effect of aggregation on the regulation of cytosolic phospholipase A₂ activity in human platelets. " Master's Thesis, University of Tennessee, 1996.
https://trace.tennessee.edu/utk_gradthes/11003