Development of anti-inflammatory drugs affecting functions of platelet adhesion molecules

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The purpose of this research project was the establishment of the basic concepts of the development of anti-inflammatory drugs affecting the cell adhesion between blood platelets and leukocytes. We got the following results.(1) The screening methods of anti-platelet drugs modifying the functions of platelet adhesion molecules have been developped, where we utilized flow cytometry with fluorescence-labeled platelets and unlabeled leukocytes (2) Recombinant soluble P-selectin inhibited the superoxide anion production in leukocytes stimulated by formyl peptides or tumor necrosis factor-alpha (TNF-alpha). However, the soluble P-selectin had no effect on the oxygen radical production in these cells stimulated by phorbor ester or arachidonic acid. (3) The Neutrophils pretreated with interleukin-8 produced the oxygen radical in response to the soluble P-selectin. (4) When monocytes derived from peripheral blood were incubated in a plate which had been coated with P-selectin, these cells produced TNF-alpha. The optimal concentration of P-selectin for the coating of the plates was found to be 0.3 mug/ml. (5) When leukocytes were incubated with immobilized P-selectin, they peoduced superoxide anion and TNF-alpha. The distribution of P-selectin ligand, sialyl Lewis X carbohydrate, on these cells was examined by immuno-fluorescence microscopy. This carbohydrate epitopes were found to form a cluster at the bottom of the cell, which is the attachment site to the plate. This observation suggested the possible relationship between the leukocyte activation by P-selectin and the cluster formation of its carbohydrate ligand.