Studies of the Nature of the Binding by Albumin of Platelet-activating Factor Released from Cells

Abstract

This report confirms that human umbilical vein endothelial cells activated by A23187 produce platelet-activating factor (PAF) (22.4 ؎ 9.9 ng/10 6 cells/h; mean ؎ S.E.). A proportion of the PAF produced (56%) was released by cells into the medium. The PAF released, however, was not detected without prior organic extraction, and the method of organic extraction was critical for detection. Extraction with 80% ethanol was not successful, but a modified methanol/chloroform extraction method was. These observations may explain some of the conflicting reports in the literature on release of PAF by activated endothelial cells. The requirements for organic extraction may reflect the nature of cell-released PAF's binding by albumin; it was observed that PAF added to identical media could be detected in a bioassay without the requirement for extraction. Such PAF was also readily degraded by PAF-acetylhydrolase added to media, while PAF released from cells was resistant to such degradation, suggesting that it was released in a "protected" configuration. Stimulation of cells was performed in media with albumin as the only extracellular macromolecule. Limited proteolytic digestion of the albumin with trypsin and pepsin showed that PAF released by cells was located exclusively between amino acids 240 and 386 (domain II), while no synthetic PAF added to media was located on this region. These results are identical to those described for the release of PAF by the early embryo. Albumin exposed to embryos had a higher thiol concentration (0.77 ؎ 0.04 mol of thiol/mol of albumin; mean ؎ S.E.) than control media to which an equivalent amount of synthetic PAF was added (0.59 ؎ 0.02 mol of thiol/mol of albumin) (measured with Ellman's reagent). Furthermore, albumin from conditioned media was more susceptible to reduction by 10 mM dithiothreitol than control albumin, as assessed by its mobility on PAGE. The protected configuration of released PAF was caused by cell-dependent conformational changes to albumin involving cysteinecysteine disulfide bonds. Partial reduction with dithiothreitol of albumin exposed to cells resulted in released PAF being able to be detected directly in a bioassay without the requirement for prior organic extraction.Platelet-activating factor (PAF 1 ; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active ether phospholipid (1, 2). It has wide tissue distribution and may function in normal physiological processes such as inflammation, neural activity, and reproduction. It may also have a role as a mediator in pathological states such as asthma, ischemia, gastric ulceration, hypertension, atherosclerosis, and shock.Many studies show that endothelial cells do not produce PAF under basal conditions in vitro, but synthesis can be stimulated by a variety of agonists (see Ref. 3 for review). PAF synthesis occurred in endothelial cells from diverse vascular beds, including aorta, umbilical vein, and pulmonary artery (4). Several reports (5-11) have shown that virtually all PAF synthesized by stimulat...

Characterization and Functional Significance of Calcium Transients in the 2-Cell Mouse Embryo Induced by an Autocrine Growth Factor

“…Mobilization of this PAF to elicit the calcium response was dependent upon extracellular protein. This is consistent with previous observations that albumin has high affinity binding sites for PAF (37), being located on domain II of albumin (27), and that extracellular albumin was essential for the release of PAF from the 2-cell embryo (26,27). Since PAF is a phospholipid it is hydrophobic, only readily entering the aqueous phase by binding to a hydrophilic carrier such as albumin.…”

The ALIAS Pilot Trial

“…18 -20 Albumin increases the production of the antiaggregatory prostaglandin, PGD2, from cyclic endoperoxides. 19 It also binds platelet-activating factor (PAF) with high affinity 21,22 and decreases PAF-induced responses in platelets. 22 Coating of thrombogenic surfaces with S-nitrosylated albumin reduces platelet adhesion and aggregation-an effect attributable both to the direct antiplatelet actions of nitric oxide and the antiadhesive properties of albumin itself.…”

Heterogeneity in the sn-1 carbon chain of platelet-activating factor glycerophospholipids determines pro- or anti-apoptotic signaling in primary neurons

“…CGNs were plated in Eagleʼs minimum essential medium (EMEM) containing 25 mM glucose, 10% FBS, 1% gentamycin, 2 mM L-glutamine, and 20 mM KCl at 37°C in a 5% CO 2 atmosphere at a density of 2 3 10 5 cells/cm 2 in 96-well plates coated with laminin (20 mg/ml) and poly-D-lysine (100 mg/ml). Cells were cultured in the serum-containing media for 72 h and washed extensively with PBS before being exposed to PAF in serumfree treatment media (EMEM, 25 mM glucose, 1% gentamycin, 2 mM L-glutamine, 20 mM KCl, and delipidated 0.025% BSA) as previously described (7,35). C 16 -PAF and C 18 -PAF (0.5-1.5 mM) were dissolved in treatment media.…”