Platelet aggregation and secretion can be inhibited by increase in the level of cyclic AMP caused by stimulation of adenylate cyclase. Endogenous agents generally act through specific receptors to stimulate the enzyme. Forskolin, from the medicinal plant Coleus forskholii, also stimulates adenylate cyclase and offers great potential for control of platelet activation. In contrast to receptor-mediated activation, forskolin acts directly on the catalytic unit or a closely associated subunit. Similarly, the inhibitor, dideoxyadenosine, interacts directly with the enzyme rather than receptors. The proposal will contribute to the long-term objective of understanding the role of adenylate cyclase in platelet responsiveness by studies on the catalytic unit. The enzyme will be characterized in terms of interaction with its substrate; with activators, such as Mn++ and forskolin; and with inhibitors such as adenosine and purine-site analogs. The possibility that the enzyme is influenced by Ca++ ions, acting through calmodulin or a Ca++ binding component of the catalytic unit, will be examined. Platelet adenylate cyclase will be solubilized using detergents and activity stabilized by ATP or forskolin. The catalytic unit will be resolved from GTP-regulatory components by gel-filtration. Attempts will be made to reconstitute the catalytic and regulatory components in terms of the ability of the regulatory component to restore responsiveness of the catalytic component to Gpp(NH)p and fluoride. The catalytic component will be further purified by a combination of dodecyl-agarose, ATP-agarose and forskolin-agarose columns, as well as appropriate conventional techniques. At each stage in the purification the responsiveness of the enzyme to activators or inhibitors will be determined. The molecular weight and subunit structure of the enzyme will also be determined at each stage, by hydrodynamic methods and by sodium dodecyl sulfate gel electrophoresis. Attempts will be made to correlate the structure with responsiveness to effectors. In particular, efforts will be made to identify and isolate a putative forskolin binding subunit and reconstitute it with the isolated catalytic unit.
| Ashby, B (1988) Cyclic AMP turnover in response to prostaglandins in intact platelets: evidence for separate stimulatory and inhibitory prostaglandin receptors. Second Messengers Phosphoproteins 12:45-57 |
| Ashby, B (1986) Kinetic evidence indicating separate stimulatory and inhibitory prostaglandin receptors on platelet membranes. J Cyclic Nucleotide Protein Phosphor Res 11:291-300 |
| Ashby, B; Kowalska, M A; Wernick, E et al. (1985) Differences in the mode of action of 1-oleoyl-2-acetyl-glycerol and phorbol ester in platelet activation. J Cyclic Nucleotide Protein Phosphor Res 10:473-83 |
