PAF-STIMULATED PHOSPHOINOSITIDE TURNOVER IN PLATELETS

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The candidate has developed an everlasting interest in membranes, phosphoinositides (PPI) and phospholipases. The immediate goal is to build a foundation for the research project outlined in this application which will enable, in the long term, to unravel the importance of PPI and phospholipases in cellular functions. Project: Breakdown of PPI by phospholipase C (PLC) in various cells, including platelets, is involved in signal transduction. The HYPOTHESIS is that (a) platelet activating factor (PAF) receptor is coupled via a G-protein to the activation and regulation of the PLC in platelets and (b)exogenous PLC or activation of endogenous PLC cause release of PI- anchored proteins from platelet membrane surfaces. There are four experimental plans. [I] TO DETERMINE MECHANISM(S) OF PAF RECEPTOR-COUPLED ACTIVATION AND DESENSITIZATION OF PLC: (A) to correlate [3H]PAF binding to PLC activation in desensitized platelets as compared to control; (B) to manipulate G-proteins by using NaF, GTPgammas and pertussis toxin and to correlate their influence on PLC activation; (C) to correlate protein kinase C-mediated phosphorylation to the activation and desensitization of PLC. [II] TO DETERMINE MOLECULAR INTERACTIONS AMONG PLC, G-PROTEINS AND PAF RECEPTOR: Platelet membranes will be solubilized and fractionated by column chromatography. [3H]PAF binding, GTPase, 32P-GTP binding and PLC activities will be monitored in fractions to establish their functional associations. [III] TO DETERMINE IMPORTANCE OF PAF RECEPTOR COUPLED ACTIVATION OF PLC IN THE HYPERSENSITIVITY OF DIABETIC HUMAN PLATELETS. [IV] TO DETERMINE THE RELEASE OF PI-ANCHORED MEMBRANE PROTEIN/GLYCOPROTEIN BY EXOGENOUS AND ENDOGENOUS PLC: Release of proteins by PLC (B. thuringiensis) from labelled platelets; identification by SDS-PAGE/fluirography and antibodies; use of differential phase partitioning to monitor PI-anchored proteins after PAF stimulation. The project will provide novel insight(s) in PAF-stimulated PPI turnover and on the importance of PI-anchored proteins in platelets.