Glycosphingolipids (GSLs) comprise a diverse class of biomolecules that are known to function as genetically-controlled recognition markers and modulators of membrane receptors and to be the targets of pathogenic agents for many cellular systems. However, very little is known about the GSls of human blood platelets. Thus, we propose, as a long-term objective, to study the pathobiochemistry of platelet GSLs. The molecular structure of all 24 platelet GSLs that we have detected by high-performance thin-layer chromatography (HPTLC) will be studied through: (1) large-scale extraction, fractionation (DEAE-Sephadex, Bio-Sil A), and purification (HPLC), (2) one-and two-dimensional proton nuclear magnetic resonance spectroscopy, (3) fast atom bombardment mass spectrometry, (4) gas chromatography. (5) microscale methylation analysis, (6) HPTLC-immunostaining, and (7) specific glycosidase treatment. GSLs with unusual structure will be used to prepare mono- clonal antibodies. The immunogenetic expression of platelet GSls will be studied by analyzing the HPTlC patterns of GSls found in individuals within a population of normal, HLA-typed donors. The function of platelet GSls will be studied by measuring their effect on ligand binding in three quantitative receptor systems. These binding systems platelet aggregation, (2) platelet serotonin uptake and binding and (3) 125I-labeled fibrinogen, fibronectin or von Willebrand factor binding to purified glycoprotein IIb/IIIa phospholipid vesicles. The role of platelet GSLs as targets for pathogenic agents will be assessed by studying the binding of patient alloantibodies, verotoxin, and E. coli to immobilized GSls using HPTLC immunostaining and HPTlC-autoradiography. The information generated through this project will contribute to a better understanding of the physiology and pathophysiology of: (1) hemostasis and thrombosis, (2) coronary artery vasospasm and hypertension, and (3) thrombocytopenias observed in patients with leukemia, infection, or undergoing bone marrow transplantation.
