It is generally accepted that activation of extravascular platelets in the lungs may contribute to the pathogenesis of many pulmonary inflammatory diseases including asthma. Although the activation of platelets has been implicated in asthma, little is known about the possible agonists responsible for their activation. We have recently reported that major basic protein (MBP), an eosinophil granule protein that is secreted when eosinophils are activated, is a potent platelet agonist. These studies also indicated that the mechanism of activation differed from that of other strong platelet agonists. It is the long- term goal of this proposal to elucidate the novel mechanism by which MBP activates human platelets. Initial studies will concentrate on confirming preliminary studies indicating that MBP does not activate phospholipase C (PLC), the first enzyme activated by most strong agonists. This will involve quantitation of diacylglycerol and inositol- 1,4,5-trisphosphate, the two second messengers, produced by the enzymatic action of PLC and examination of the effect of the PLC inhibitor, U- 73,122, on MBP-mediated platelet activation. Next, studies measuring calcium influx with 45Ca and others using inhibitors of calcium influx will be performed to test the hypothesis that the slow rise in intracellular calcium seen in MBP activated platelets is due to extracellular influx rather than release from intracellular stores. Despite the absence of significant levels of the second messengers normally associated with the activation of protein kinases, the normal phosphorylation of both pleckstrin and myosin light chain in MBP- stimulated platelets clearly indicates that protein kinases are activated by MBP. The role of unsaturated fatty acids in the activation of an isoenzyme of protein kinase C will be examined using both radiotracer techniques and mass measurements of the fatty acids formed during MBP- mediated platelet activation. The role of protein tyrosine kinases will also be investigated. The induction of protein tyrosine phosphorylation will be measured by an immunoblotting with an anti-phosphotyrosine antibody. Finally, the functional significance of protein tyrosine phosphorylation will be studied by examining the effects of the protein tyrosine kinase inhibitor, genistein, on the various steps in signal transduction.
| Plager, Douglas A; Davis, Mark D P; Andrews, Amy G et al. (2009) Eosinophil ribonucleases and their cutaneous lesion-forming activity. J Immunol 183:4013-20 |
