Human blood cells possess a unique array of lipoxygenase enzymes. Polymorphonuclear leukocytes contain a 5-lipoxygenase which is able to metabolize arachidonic acid to the biologically important mediators, such as 5-H(P)ETE and leukotriene B4. Reticulocytes possess a 15lipoxygenase which is believed to be involved in the triggering of intracellular organelle degradation prior to erythrocyte maturation. Platelets express 12-lipoxygenase which metabolizes arachidonic acid to 12-hydro(pero)xy-eicosatetraenoic acid (12-H(P)ETE). Whereas the biological roles of the first two lipoxygenases are reasonably well established, the function of platelet 12-lipoxygenase remains unsettled despite several postulated activities of 12-H(P)ETE. It is the aim of the present proposal to isolate the molecular tools necessary to elucidate further the role of 12-H(P)ETE in human biology. In the first specific aim complementary DNA (cDNA) clones encoding human platelet 12-lipoxygenase will be isolated by a polymerase chain reaction (PCR) cloning approach. Extra-platelet expression of 12-lipoxygenase will be investigated by Northern analysis and putative isoforms characterized by PCR cloning. In the second specific aim structure/functional properties of the lipoxygenases will be studied through in vitro mutagenesis and chimeric lipoxygenase expression studies in transfected COS-M6 cells. The potential role and mechanism of intracellular translocation in the activation of 12-lipoxygenase will be investigated. In the third specific aim growth factors, steroids and various platelet constituents and the phorbol ester (PMA) will be examined for their effects on the regulation of 12-lipoxygenase gene expression through Northern analysis studies. Finally, in the fourth specific aim the 12lipoxygenase gene will be characterized through analysis of genomic clones and compared to the 5-lipoxygenase gene. The chromosomal localization of all 3 lipoxygenases will be determined to see if the lipoxygenase gene family is linked to one chromosomal segment. Information obtained from these studies will elucidate our knowledge of the lipoxygenases and ultimately lead to an understanding of 12-H(P)ETE's biological role.
