It has been suggested that the hydrolysis of phosphatidylinositol (PI) has a gating function in a mechanism whereby the activation of receptors at cell surfaces induces the mobilization of Ca++. Human platelets have been shown to contain a PI-specific phospholipase C (PI-PLC) which is activated by exposure of platelets to thrombin. They also contain Alpha-adrenergic receptors which, upon stimulation, lead to Ca++ transport across the plasma membrane, enhanced synthesis of PI, and depressed adenylate cyclase activity. We propose to determine 1) whether hydrolysis of phosphoinositides occurs in platelets activated by epinephrine under a variety of conditions, and whether the circumstances controlling PI metabolism in such platelets are consistent with a Ca++-gating role for PI turnover, 2) the effects of cholesterol enrichment/depletion or treatment with PI-PLC upon the activity of platelet membrane adenylate cyclase, 3) whether a polyphosphoinositide-specific PLC is present in platelet membranes, 4) the effects of other phospholipids and fatty acid on the activity of soluble PI-PLC, and 5) the extent of potentiation by epinephrine or the adenylate cyclase inhibitor SQ22536 of collagen, ADP, and low-dose thrombin-induced hydrolysis of platelet phospholipid. For most of these studies, we will employ techniques of lipid resolution with which we are familiar, including TLC, HPLC, and GC. Platelets will be loaded with or depleted of cholesterol using cholesterol-rich or -poor phospholipid vesicles, and their membranes isolated by a method now used routinely in this laboratory, which yields preparations whose adenylate cyclase is responsive to epinephrine and PGD2. Turnover of PI will be assessed with the aid of both radioisotopic labeling and mass determinations. The clarification of the role of Alpha-adrenergic stimulation, particularly as a synergistic cohort for other agonists and as an event already shown to be especially susceptible to modification by cholesterol, should enhance our understanding of the events controlling platelet aggregation in normal and pathological states such as thrombosis.
| Rittenhouse, S E (1987) Measurement of capillary gas chromatography of mass changes in myo-inositol trisphosphate. Methods Enzymol 141:143-9 |
| Banga, H S; Simons, E R; Brass, L F et al. (1986) Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A 83:9197-201 |
| Brass, L F; Laposata, M; Banga, H S et al. (1986) Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhi J Biol Chem 261:16838-47 |
