There is good reason to suspect that both platelet-activating factor and thromboxane A2 are involved in propagating the thrombotic response of platelets. Only by fully understanding the mechanism of their formation will it be possible to rationally develop drugs to inhibit the biosynthesis of these biologically-active lipids. Although aspirin inhibits the formation of thromboxane A2, it does not inhibit the formation of products of the lipoxygenase pathway or platelet-activating factor. Presently no drugs are known which inhibit the biosynthesis of this latter compound. This application has two major aims: 1. To determine which individual molecular species of phospholipid are hydrolysed during platelet stimulation by thrombin, collagen and the calcium ionophore, ionomycin to release arachindonic acid for thromboxane formation and 1-0-alkyl-1-lyso-sn-glycerophosphocholine for the formation of platelet-activating factor. An associated goal will be to use an intracellular fluorescent probe called Quin-2 to determine the intracellular increases in free calcium that accompany the hydrolysis of individual phospholipid species. 2. To determine the distribution of individual molecular species of phospholipid in the platelet plasma membrane and in its organelles, and to ascertain how these distributions are changed during stimulation of platelets. To acheive these aims we will rely heavily on elegant new separations afforded by high-performance liquid chromatography (HPLC).
