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Masters Theses
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 pl25FAK (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 pl25FAK, 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