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Characterization of a platelet activating factor using forskolin and methylcarbamyl PAF

University of British Columbia

Platelet activating factor is an important mediator of septic shock and inflammation. The action of PAF on target cells is mediated through specific membrane bound receptors. PAF binding to receptors initiates a cascade of biochemical signalling events leading to the aggregation of platelets or the activation of other target cells. In this work we characterized the PAF-receptor physiochemical properties in platelets and investigated its interaction with signalling systems. The effect of forskolin on PAF receptor binding was studied. Studies were also conducted towards elucidation of the nature of the PAF receptor. In this direction the binding of the stable PAP analog l-0-hexadecyl-2-O-(methylcarbamyl)-sn-glycero- 3-phosphocholine (MC-PAF), a compound with potential uses in PAF receptor purification and antibody production was evaluated. Forskolin is commonly used in the investigation of the effects of stimulated adenylyl cyclase activity in cells. In the first project the effect of forskolin on platelet activating factor receptor was explored. Rabbit platelets treated with forskolin prior to PAP binding resulted in a 30-40% decrease in binding which translated to a change in receptor number on the cell surface. This decrease in PAF binding caused by forskolin was concomitant with a decrease in platelets’ physiological response to PAP. However, the forskolin induced decrease in PAF binding was not a consequence of cAMP formation as the addition of a cAMP analog could not mimic the actions of forskolin. Additionally, the inactive analog of forskolin, dideoxyforskolin, which does not activate adenylyl cyclase, also reduced PAF binding to its receptor. Reduction of PAP binding by forskolin and dideoxyforskolin was also demonstrated in isolated platelet membranes. The action of forskolin was also found to be independent of G-protein involvement. We therefore concluded the PAF receptor may be regulated by several factors that are triggered by forskolin. These elements are independent of adenylyl cyclase involvement. In the second project MC-PAF was evaluated using rabbit platelets. MC-PAF was found to be approximately 3-5 fold less active in causing platelet aggregation and serotonin release. Aggregation caused by MC-PAF was completely inhibited by WEB 2086, a PAF receptor antagonist. Ligand binding studies revealed that MC-PAF binds with approximately two-fold lower affinity than PAF to the PAF receptor (Kd=l .47 and 0.62 nM for MC-PAF and PAF, respectively). Studies in anesthetised rats showed that both PAF and MC-PAP caused a similar reduction in blood pressure without changes in either heart rate or EKG parameters. The susceptibility of PAF and MC-PAF to hydrolysis by serum acetyihydrolases was investigated. PAP was found to be fully hydrolyzed after 5 mm in rabbit serum while MC-PAF was not degraded significantly after a 1 hour exposure. These studies suggested that MC-PAF is a full agonist of the PAF receptor, both in vitro and in vivo, and is more stable than PAP against serum acetylhydrolases, thus making MC-PAF a useful compound for purifying the PAF receptor as well as raising PAF antibodies.

Thesis/Dissertation

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2008-12-16

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http://hdl.handle.net/2429/2976

Experimental Medicine

University of British Columbia

UBCV

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