Date of Award

1-1993

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Richard J. Haslam

Abstract

Comparative studies on the regulation of secretion from
dense and α-granules of electropermeabilized human platelets
indicated the involvement of three distinct factors, Ca²⁺, protein
kinase C (PKC) and an unidentified GTP-binding protein (GE). Any
two of these factors must be present for marked secretion from
dense or α-granules to occur. Thus, Ca²⁺ was not essential to the
exocytotic mechanism and the combination of phorbol ester (a PKC
activator) and a metabolically-stable GTP analogue (GTP[S]) could
produce marked Ca²⁺-independent secretory response. Ca²⁺ appeared
to have a more modulatory role, enhancing both the rate and extent
of secretion. Secretion did not correlate with phospholipase C (PLC) activity or with the accumulation of 1,2-diacylglycerol (DAG), both of which required Ca²⁺ and were inhibited by phorbol ester. The Ca²⁺-independent activation of phospholipase A₂ was shown for the first time in platelets, but moderate to complete inhibition of this enzyme had no effect on secretion or other hospholipase activities. Only the activation of phospholipase D, assayed by the formation of [³H]phosphatidic acid (PA) and
[³H]phosphatidyl-ethanol (PEt), in the absence and presence of ethanol, respectively, correlated with secretion under all
conditions tested. BAPTA and analogues, known inhibitors of Ca²⁺--independent secretion in other permeabilized cells, caused parallel dose-dependent inhibitions of PLD activity and secretion. PKC activity detected by the phosphorylation of its major endogenous substrate, pleckstrin, was enhanced by GTP[S] apparently by stimulating the formation of PA, as well as of DAG in the presence of Ca²⁺. An optimal dose of the protein kinase inhibitor staurosporine could not block secretion produced by GTP[S] in the presence of Ca²⁺, suggesting an additional role for PLD activation, independent of protein phosphorylation. However, although correlations between PLD activity and secretion were also been in intact platelets, this enzyme is known to account for only 10 - 20% of the total PA formed in intact thrombin-stimulated platelets .. This PA may be different from that formed via the
PLC - DAG kinase pathway and therefore the nature and subcellular
localization of this PA will have to be investigated in order to
establish whether PLD has a major role in secretion. The possible
existence of an alternate GTP[S]-stimulated effector protein
cannot be excluded.


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