title: Endotoxic lipid A interaction with human platelets. Structure-function analysis of lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide.
abstract: We previously reported that human blood platelets are directly stimulated by endotoxic Lipid A via the protein kinase C pathway (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). To study the relationship between the molecular structure of Lipid A and its ability to activate human platelets, we used Lipid A homologs derived from Salmonella minnesota Re595 lipopolysaccharide. Preparations of Lipid A are heterogeneous in regard to the degree of substitution of fatty acids which result in multiple homologs. These were separated by thin-layer chromatography and characterized by fast atom bombardment spectroscopy and related techniques (Johnson R. S., Her, G.-R., Grabarek, J., Hawiger, J., and Reinhold, V. N. (1990) J. Biol. Chem. 265, 8108-8116). The homologs of monophosphoryl Lipid A (MLA) present in fractions TLC-8 (heptaacyl MLA ion, m/z 1953), TLC-7 (three hexaacyl species with predominant MLA ion m/z 1715), and TLC-6 (four pentaacyl homologs with predominant MLA ion, m/z 1505) induced secretion of [14C]serotonin and aggregation of platelets. Lipid A homologs in fractions TLC-5 (three tetraacyl MLA ions, m/z 1323, 1307, and 1279), TLC-4 (one major triacyl MLA ion, m/z 1097), TLC-3 (tetraacyl MLA ion, m/z 1278), TLC-2 (a diphosphoryl hexaacyl Lipid A ion, m/z 1795, and several ions of low abundance), and TLC-1 (two ions, m/z 1097 and 666) were not active in regard to human platelet aggregation and [14C]serotonin secretion. The most active homolog was heptaacyl MLA ion, m/z 1953, present in TLC-8, while homologs present in TLC-7 and TLC-6 were 5 and 10 times less active, respectively. Rapid phosphorylation of a human platelet protein of Mr 40,000-47,000 (P47), a substrate for protein kinase C activation, preceded secretion of serotonin when platelets were triggered by the most active heptaacyl MLA ion, m/z 1953. These events were time-dependent, with half-maximal response of phosphorylation of P47 at 30 s and [14C]serotonin secretion at 45 s. A marked difference in the degree of phosphorylation of P47 was observed with heptaacyl MLA homolog present in TLC-8 inducing complete phosphorylation (97%), whereas less acylated Lipid A homologs present in TLC-1 caused marginal phosphorylation (20%). These results indicate that the degree of acylation of monophosphoryl Lipid A determines its functional properties toward human platelets in regard to secretion of [14C]serotonin, aggregation, and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215.
authors.forename: J
authors.initials: J
authors.lastname: Grabarek
authors.forename: G R
authors.initials: GR
authors.lastname: Her
authors.forename: V N
authors.initials: VN
authors.lastname: Reinhold
authors.forename: J
authors.initials: J
authors.lastname: Hawiger
mesh_terms: D000215:Acylation; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008050:Lipid A / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000032:analysis*; D013058:Mass Spectrometry; D008970:Molecular Weight; D010710:Phosphates; D010750:Phosphoproteins / Q000097:blood; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D012475:Salmonella / Q000032:analysis*; D012701:Serotonin / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D008050:Lipid A; D008070:Lipopolysaccharides; D010710:Phosphates; D010750:Phosphoproteins; D012701:Serotonin; D011493:Protein Kinase C