<p>Comparative studies on the regulation of secretion from<br />dense and α-granules of electropermeabilized human platelets<br />indicated the involvement of three distinct factors, Ca²⁺, protein<br />kinase C (PKC) and an unidentified GTP-binding protein (GE). Any<br />two of these factors must be present for marked secretion from<br />dense or α-granules to occur. Thus, Ca²⁺ was not essential to the<br />exocytotic mechanism and the combination of phorbol ester (a PKC<br />activator) and a metabolically-stable GTP analogue (GTP[S]) could<br />produce marked Ca²⁺-independent secretory response. Ca²⁺ appeared<br />to have a more modulatory role, enhancing both the rate and extent<br />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<br />[³H]phosphatidyl-ethanol (PEt), in the absence and presence of ethanol, respectively, correlated with secretion under all<br />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<br />PLC - DAG kinase pathway and therefore the nature and subcellular<br />localization of this PA will have to be investigated in order to<br />establish whether PLD has a major role in secretion. The possible<br />existence of an alternate GTP[S]-stimulated effector protein<br />cannot be excluded.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/8770 |
| Date | January 1993 |
| Creators | Coorssen, Jens R. |
| Contributors | Haslam, Richard J., Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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