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• W2024587005 abstract "This report confirms that human umbilical vein endothelial cells activated by A23187 produce platelet-activating factor (PAF) (22.4 ± 9.9 ng/106 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 μmol of thiol/μmol of albumin; mean ± S.E.) than control media to which an equivalent amount of synthetic PAF was added (0.59 ± 0.02 μmol of thiol/μmol 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 cysteine-cysteine 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. This report confirms that human umbilical vein endothelial cells activated by A23187 produce platelet-activating factor (PAF) (22.4 ± 9.9 ng/106 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 μmol of thiol/μmol of albumin; mean ± S.E.) than control media to which an equivalent amount of synthetic PAF was added (0.59 ± 0.02 μmol of thiol/μmol 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 cysteine-cysteine 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 1The abbreviations used are: PAF, platelet-activating factor; PAF:AH, PAF-acetylhydrolase; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; BSA, bovine serum albumin; ECCM, endothelial cell-conditioned medium; ECM, embryo-conditioned medium; HTFM, human tubal fluid medium; RIA, radioimmunoassay; ANOVA, analysis of variance. 1The abbreviations used are: PAF, platelet-activating factor; PAF:AH, PAF-acetylhydrolase; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; BSA, bovine serum albumin; ECCM, endothelial cell-conditioned medium; ECM, embryo-conditioned medium; HTFM, human tubal fluid medium; RIA, radioimmunoassay; ANOVA, analysis of variance.; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active ether phospholipid (1Dempoulos C.A. Pinckard R.N. Hanahan D.J. J. Biol. Chem. 1979; 254: 9355-9358Abstract Full Text PDF PubMed Google Scholar, 2Benveniste J. Tence M. Varenne P. Bidault J. Boullet C. Polonsky P. C. R. Seances Acad. Sci. 1979; 289: 1037-1040PubMed Google Scholar). 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. 3Bratton D. Henson P.M. Barnes P.J. Page C.P. Henson P.M. Platelet Activating Factor and Human Disease. Blackwell Scientific Publications, Oxford1989: 23-57Google Scholar for review). PAF synthesis occurred in endothelial cells from diverse vascular beds, including aorta, umbilical vein, and pulmonary artery (4Whatley R.E. Nelson P. Zimmerman G.A. Stevens D.L. Parker C.J. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1988; 264: 6325-6333Abstract Full Text PDF Google Scholar). Several reports (5Prescott S.M. Zimmerman G.A. McIntyre T.M. Proc. Natl. Acad. Sci. 1984; 81: 3534-3538Crossref PubMed Scopus (338) Google Scholar, 6McIntyre T.M. Zimmerman G.A. Satoh K. Prescott S.M. J. Clin. Invest. 1985; 76: 271-280Crossref PubMed Scopus (313) Google Scholar, 7Zimmerman G.A. McIntyre T.M. Prescott S.M. Circulation. 1985; 72: 718-727Crossref PubMed Scopus (78) Google Scholar, 8Hirafugi M. Mencia-Heurta J.M. Benveniste J. Biochim. Biophys. Acta. 1987; 930: 359-369Crossref PubMed Scopus (30) Google Scholar, 9Zimmerman G.A. Whatley R.E. McIntyre T.M. Prescott S.M. Am. Rev. Respir. Dis. 1987; 136: 204-207Crossref PubMed Scopus (19) Google Scholar, 10Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Br. J. Pharmacol. 1989; 96: 503-505Crossref PubMed Scopus (23) Google Scholar, 11Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3215-3219Crossref PubMed Scopus (101) Google Scholar) have shown that virtually all PAF synthesized by stimulated endothelial cells remained associated with the cells. By contrast, a number of reports (12Camussi G. Aglietta M. Malavasi F. Tetta C. Piacibello W. Sanavio F. Bussolino F. J. Immunol. 1983; 131: 2397-2403PubMed Google Scholar, 13Bussolino F. Brevario F. Tetta M. Aglietta M. Mantovani A. Dejana E. J. Clin. Invest. 1986; 77: 2027-2033Crossref PubMed Scopus (173) Google Scholar, 14Bussolino F. Brevario F. Aglietta M. Sanavio F. Bosia A. Dejana E. Biochim. Biophys. Acta. 1987; 927: 43-54Crossref PubMed Scopus (20) Google Scholar, 15Camussi G. Bussolino F. Salvidio G. Baglioni C. J. Exp. Med. 1987; 166: 1390-1404Crossref PubMed Scopus (285) Google Scholar, 16Bussolino F. Camussi G. Baglioni C. J. Biol. Chem. 1988; 263: 11856-11861Abstract Full Text PDF PubMed Google Scholar, 17Holtzman M.J. Ferdman B. Bohrer A. Turk J. Biochem. Biophys. Res. Commun. 1991; 177: 357-364Crossref PubMed Scopus (19) Google Scholar, 18Cabre F. Tost D. Suesa N. Gutierrez P. Ucedo P. Mauleon D. Carganicco G. Agents & Actions. 1993; 38: 212-219Crossref PubMed Scopus (14) Google Scholar, 19Montrucchio G. Bergerone S. Bussolino F. Alloatti G. Silvestro L. Lupia Cravetto A. DiLeo M. Emanuelli G. Camussi G. Circulation. 1993; 88: 1479-1483Crossref Scopus (42) Google Scholar) show that as much as 25% was released.A common feature of reports that have failed to detect PAF release from endothelial cells (5Prescott S.M. Zimmerman G.A. McIntyre T.M. Proc. Natl. Acad. Sci. 1984; 81: 3534-3538Crossref PubMed Scopus (338) Google Scholar, 6McIntyre T.M. Zimmerman G.A. Satoh K. Prescott S.M. J. Clin. Invest. 1985; 76: 271-280Crossref PubMed Scopus (313) Google Scholar, 7Zimmerman G.A. McIntyre T.M. Prescott S.M. Circulation. 1985; 72: 718-727Crossref PubMed Scopus (78) Google Scholar, 8Hirafugi M. Mencia-Heurta J.M. Benveniste J. Biochim. Biophys. Acta. 1987; 930: 359-369Crossref PubMed Scopus (30) Google Scholar, 9Zimmerman G.A. Whatley R.E. McIntyre T.M. Prescott S.M. Am. Rev. Respir. Dis. 1987; 136: 204-207Crossref PubMed Scopus (19) Google Scholar, 10Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Br. J. Pharmacol. 1989; 96: 503-505Crossref PubMed Scopus (23) Google Scholar, 11Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3215-3219Crossref PubMed Scopus (101) Google Scholar) is that they have used methods other than a modified chloroform-methanol extraction method (20Pinckard R.N. Farr R.S. Hanahan D.J. J. Immunol. 1979; 123: 1847-1857PubMed Google Scholar) for extracting phospholipids prior to assay. A recent study (21Ammit A.J. O'Neill C. J. Reprod. Fertil. 1997; 109: 309-318Crossref PubMed Scopus (25) Google Scholar) showed that this method of extracting media was required to detect PAF released from preimplantation embryos, due apparently to PAF's exclusive binding to domain II of albumin. Binding of PAF at that site on albumin resulted in PAF being protected from the actions of PAF-acetylhydrolase (PAF:AH) and also resulted in PAF being undetectable in bioassays without prior extraction. It was suggested that binding of PAF to domain II of albumin resulted in it being presented in a protected form (21Ammit A.J. O'Neill C. J. Reprod. Fertil. 1997; 109: 309-318Crossref PubMed Scopus (25) Google Scholar). PAF added to medium also bound to albumin but was not found on domain II and was not in a protected form. We were interested to determine whether binding of PAF to albumin in this protected configuration occurs for PAF released by cells other than embryos and also to investigate the nature of the interaction which results in the protected configuration.In this study we show that activation of endothelial cells results in the production of PAF and its release into medium. It is bound to albumin in a protected form, being exclusively located on domain II of albumin. It is also shown that binding of embryo-derived and endothelial cell-derived PAF to albumin was associated with conformational changes to albumin which involved modification of the cysteine-cysteine disulfide bonds, while the the conformation conferred by disulfide bonds was necessary for PAF to remain in its “protected” configuration.DISCUSSIONThe results confirm that human umbilical vein endothelial cells activated by A23187 produce PAF. While a significant proportion of the PAF remained associated with the cells, much of it was released into medium. This PAF was not bioactive in media in a platelet aggregation assay, nor was it recovered following ethanolic extraction. This behavior of endothelial cell-derived PAF contrasts with that of synthetic PAF added to media under the same conditions.Synthetic PAF was readily detected by platelet aggregation without the requirement for extraction, and was also readily extracted by ethanol (80%). We have shown previously (21Ammit A.J. O'Neill C. J. Reprod. Fertil. 1997; 109: 309-318Crossref PubMed Scopus (25) Google Scholar) that this behavior of synthetic PAF is independent of the method of its preparation. Cell-released PAF was also apparently different from PAF retained by cells, which can be readily extracted by 80% ethanol treatment (11Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3215-3219Crossref PubMed Scopus (101) Google Scholar). The reason why the modified Bligh-Dyer method was able to extract PAF from ECCM but 80% ethanol could not is not clear. The relative volume of methanol used was much greater, being 1:19 (media:methanol) compared with only 1:4 (media:ethanol). We have found that the very slow dropwise addition of media to the larger volume of methanol followed by incubation at room temperature is required for successful extraction. The subsequent secondary extraction of methanol with chloroform may also be important in the successful recovery of PAF. Systematic study of the variables is needed to define the minimum requirements for successful extraction of PAF from media released from cells.The failure of previous reports to detect the release of PAF from endothelial cells may have been due to the measurement of PAF without extraction, or following simple ethanolic extraction procedures, or using various other mixtures of chloroform and methanol (5Prescott S.M. Zimmerman G.A. McIntyre T.M. Proc. Natl. Acad. Sci. 1984; 81: 3534-3538Crossref PubMed Scopus (338) Google Scholar, 6McIntyre T.M. Zimmerman G.A. Satoh K. Prescott S.M. J. Clin. Invest. 1985; 76: 271-280Crossref PubMed Scopus (313) Google Scholar, 7Zimmerman G.A. McIntyre T.M. Prescott S.M. Circulation. 1985; 72: 718-727Crossref PubMed Scopus (78) Google Scholar, 8Hirafugi M. Mencia-Heurta J.M. Benveniste J. Biochim. Biophys. Acta. 1987; 930: 359-369Crossref PubMed Scopus (30) Google Scholar, 9Zimmerman G.A. Whatley R.E. McIntyre T.M. Prescott S.M. Am. Rev. Respir. Dis. 1987; 136: 204-207Crossref PubMed Scopus (19) Google Scholar, 10Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Br. J. Pharmacol. 1989; 96: 503-505Crossref PubMed Scopus (23) Google Scholar, 11Stewart A.G. Dubbin P.N. Harris T. Dusting G.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3215-3219Crossref PubMed Scopus (101) Google Scholar, 12Camussi G. Aglietta M. Malavasi F. Tetta C. Piacibello W. Sanavio F. Bussolino F. J. Immunol. 1983; 131: 2397-2403PubMed Google Scholar). It can be concluded that extraction of cell-conditioned media with the methods used in this report are required before release of PAF by cells can be excluded. It also shows that recovery of synthetic PAF added to media does not act as a suitable positive control for the recovery of cell-released PAF.One study (37Whatley R.E. Clay K.L. Chilton F.H. Triggiani M. Zimmerman G.A. McIntyre J.M. Prescott S.M. Prostaglandins. 1992; 432: 21-29Crossref Scopus (28) Google Scholar) showed that stimulated endothelial cells produce both PAF and its sn-1-acyl analogue (acyl-PAF), with acyl-PAF being the predominant product. That study measured the concentration of PAF and acyl-PAF remaining associated with the cells but did not determine whether either was released into medium. It was suggested that acyl-PAF can mimic the biological actions of PAF, but was substantially less potent. Thus measurement of the bioactive material released by endothelial cells does not exclude the possibility that it was induced by acyl-PAF. The antibody used in the RIA in the current study is more selective for PAF (38Smal M.A. Baldo B.A. Harle D.G. J. Mol. Recognit. 1990; 3: 168-172Crossref Scopus (3) Google Scholar) than that used in earlier studies (39Sugatani J. Lee D.Y. Hughes K.T. Saito K. Life Sci. 1990; 46: 1443-1450Crossref PubMed Scopus (25) Google Scholar). The good agreement on the amount of PAF released by cells detected by the bioassay and the RIA is unlikely if the bioactivity was due to acyl-PAF. The amount of released PAF detected by the RIA was similar after treatment of extracted material with phospholipase A1 (results not shown). Since acyl-PAF, but not PAF, is sensitive to digestion with phospholipase A1 (37Whatley R.E. Clay K.L. Chilton F.H. Triggiani M. Zimmerman G.A. McIntyre J.M. Prescott S.M. Prostaglandins. 1992; 432: 21-29Crossref Scopus (28) Google Scholar), we conclude that most of the bioactivity detected from endothelial cells was PAF. This does not exclude the possibility that acyl-PAF may also be released and bind in a similar fashion to PAF. This question could be addressed by mass spectrometric analysis of the released phospholipids.It has previously been demonstrated that extracellular albumin is an acceptor for PAF released by cells (40Ludwig J.C. Hoppens C. McManus L.M. Mott G.E. Pinckard R.N. Arch. Biochem. Biophys. 1985; 241: 337-347Crossref PubMed Scopus (48) Google Scholar), presumably by removing PAF from the lipophilic environment of the membrane. We observed (21Ammit A.J. O'Neill C. J. Reprod. Fertil. 1997; 109: 309-318Crossref PubMed Scopus (25) Google Scholar) that the release of PAF by preimplantation embryos was exclusively at domain II of albumin, and the current study shows that this is also the binding site for PAF released by endothelial cells.BSA (583 amino acids) has 80% sequence homology with human serum albumin (41Brown J.R. Fed. Proc. 1974; 33: 1389Google Scholar), and most structural studies have been performed on human serum albumin. It is a “heart-shaped” globular molecule, similar to an equilateral triangle. It is proposed that there are three cylindrical peptide segments (domains I, II, and III) connected by solvent exposed α-helical chains, which are common sites of proteolytic cleavage. Domain II contains a hydrophobic core, which can provide a binding region for hydrophobic molecules (42Brodersen R. Andersen S. Vorium H. Nielsen S.U. Pedersen A.O. Eur. J. Biochem. 1990; 189: 339-349Crossref Scopus (68) Google Scholar, 43Brown J.R. Shockley P. Jost P.C. Griffith O.H. Lipid-Protein Interactions. 1. John Wiley & Sons, New York1982: 25-68Google Scholar). Clayet al. (44Clay K.L. Johnson C. Henson P. Biochim. Biophys. Acta. 1990; 1046: 309-314Crossref PubMed Scopus (35) Google Scholar) provided kinetic evidence that albumin possessed four binding sites for PAF which had an average dissociation constant of 0.1 μm. The rabbit PAF receptor has an estimatedk d for PAF of ∼0.5 nm (45Dent G. Ukena D. Barnes P.J. Barnes P.J. Page C.P. Henson P.M. Platelet Activating Factor and Human Disease. Blackwell Scientific Publications, Oxford1990: 23-57Google Scholar). Synthetic PAF added to control culture medium caused platelet aggregation in whole blood down to a concentration of 0.5 ng/ml (∼0.9 nm). However, at a concentration of 3.0 ng/ml (∼5.6 nm), endothelial cell-derived PAF in untreated culture medium did not cause platelet activation. This concentration was well below the stated k d for PAF binding by albumin, yet above the k d of the platelet receptor. It might therefore be expected that the kinetics would favor PAF transfer from albumin to the PAF-receptor, as was the case for synthetic PAF. The absence of this suggests that PAF released by cells either binds to sites on albumin that have a much higher affinity than those described by Clay et al. (44Clay K.L. Johnson C. Henson P. Biochim. Biophys. Acta. 1990; 1046: 309-314Crossref PubMed Scopus (35) Google Scholar), or that the conformation of albumin causes PAF bound at domain II to be solvent- or sterically protected in a way that does not occur for PAF binding to albumin in the absence of cells.The secondary and tertiary structure of albumin is highly dependent on the presence of 17 interchain disulfide bonds, which link 34 (of the available 35) cysteine residues. The free cysteine residue is generally at amino acid 34 in domain I of albumin. This should be detected as 1 μmol of thiol/μmol of BSA, yet approximately 0.5 μmol of thiol/μmol of albumin is normally detected (32Janatova J. Fuller J.K. Hunter M.J. J. Biol. Chem. 1968; 243: 3612-3622Abstract Full Text PDF PubMed Google Scholar, 46Ghiggeri G.M. Candiano G. Delfino G. Querirolo C. Clin. Chim. Acta. 1983; 130: 257-261Crossref PubMed Scopus (7) Google Scholar). This may be the result of dimerization of albumin (47King T.P. J. Biol. Chem. 1961; 236: PC5Abstract Full Text PDF PubMed Google Scholar) in solution or be due to the reactive Cys-34 being “solvent-protected” by the helices in some albumin molecules (48Carter D.C. He X.-M. Munson S.H. Twigg P.D. Gernert K.M. Broom M.B. Miller T.Y. Science. 1989; 244: 1195-1198Crossref PubMed Scopus (532) Google Scholar). BSA exposed to embryos expressed more reactive thiol residues than untreated medium with PAF added, showing that binding of PAF per se did not induce this increase in thiol concentration. The increase in thiol concentration was therefore apparently due to a cell-dependent process. The observation that albumin was more readily reduced by dithiothreitol after incubation with embryos suggests that the cell-dependent process involved conformational changes to albumin involving cysteine-cysteine disulfide bonds. It has been shown (49Reed R.G. Burrington C.M. J. Biol. Chem. 1989; 264: 9867-9872Abstract Full Text PDF PubMed Google Scholar) that interaction of albumin with cells or surfaces caused a conformational change resulting in a mixed population of albumin molecules. While the mechanisms of the change are not well understood, it appears to cause a flattening of the molecule giving it a greater surface area when bound to cells and a higher binding affinity. It has been proposed (49Reed R.G. Burrington C.M. J. Biol. Chem. 1989; 264: 9867-9872Abstract Full Text PDF PubMed Google Scholar) that this conformational change promotes dissociation of passenger fatty acids, facilitating transfer from albumin to the cell. It will be of interest to determine whether a similar mechanism operates in reverse for the removal of PAF from cells.Exposure of embryo-conditioned media and endothelial cell-conditioned media to dithiothreitol, under conditions expected to cause reduction of albumin, resulted in a large proportion of the expected PAF activity present to be detected in a direct assay of platelet aggregation (without prior organic extraction). Control experiments confirmed that the aggregation was not caused by the dithiothreitol itself, nor did dithiothreitol reduce the sensitivity of the assay to PAF. This result suggests that binding of PAF to domain II of albumin, which results in its protected configuration, involves protein conformation that is dependent upon the disulfide bonds that can be reduced by dithiothreitol.The observation that PAF activity was lost with prolonged exposure to dithiothreitol may have several possible explanations. One is that as albumin was reduced (and thus changed conformation) it lost its affinity for PAF, resulting in PAF being adsorbed by the surfaces of the holding tube, as is known to occur in protein-free media (50Benveniste J. Henson P.M. Cochrane C.G. J. Exp. Med. 1972; 136: 1356-1377Crossref PubMed Scopus (964) Google Scholar). Using different types of vessels may reduce this loss of activity. Confirmation that the cause of the time-dependent loss of activity was its loss to hydrophobic surfaces, was the ability to recover PAF from XAD-2 chromatography beads after incubation with dithiothreitol treated ECCM. The results indicate that as the conformation of albumin is altered with reduction, PAF becomes “exposed” and hence available to bind to the platelet receptor, causing platelet aggregation in the bioassay. As reduction proceeds, PAF seems to be readily lost from albumin and bound by other hydrophobic surfaces, such as the test tube or XAD-2 beads.These experiments provide indirect evidence that the protected nature of PAF's binding to albumin involves disulfide bonds between cysteine molecules. The observation that breaking these disulfide bonds is necessary for making PAF accessible in vitro infers that some form of disulfide isomerization or reduction may be required for PAF's “release” from the cell and binding on domain II of albumin. The changed thiol status of albumin following exposure to cells implicates cellular enzymes in this process.Studies with embryo-conditioned medium show that, while embryo-derived PAF in untreated media is inactive in vitro (21Ammit A.J. O'Neill C. J. Reprod. Fertil. 1997; 109: 309-318Crossref PubMed Scopus (25) Google Scholar), upon injection into animals it can induce thrombocytopenia (51O'Neill C. J. Reprod. Fertil. 1985; 75: 375-380Crossref PubMed Scopus (123) Google Scholar). Such studies show that cellular-dependent factors must be involved in the creation and processing of cell-released PAF in its protected form and its exposure in vivo to allow it to be bioactive. Further experiments are required to determine the nature of this mechanism. One possibility may be the presence of cell surface protein disulfide isomerases (52Rubartelli A. Bajetto A. Allavena G. Wollman E. Sitia R. J. Biol. Chem. 1992; 267: 24161-24164Abstract Full Text PDF PubMed Google Scholar, 53Dean M.F. Martin H. Sansom P.A. Biochem. J. 1994; 304: 861-867Crossref PubMed Scopus (12) Google Scholar), which might cause reduction of some disulfides resulting in solvent exposure of domain II, facilitating the loading of PAF at this site. It might be speculated that a similar process, in reverse, may be required to allow PAF to be made available at target cells.While there is some controversy regarding the ability of some cell types to release PAF, for some other cell types such as activated basophils, there is general agreement that PAF is released upon cellular activation. This might suggest that PAF released from some cell types, or under some conditions, is not in the protected configuration described in this report. Systematic investigations are required to assess this possibility.This study used defined culture media with albumin as the only extracellular macromolecule. The next important question to investigate will be to determine whether PAF binds to albumin in this protected form when a complex protein source such as serum is present. Several studies have detected PAF in blood (54Caramelo C. Fernandez-Gallardo S. Marin C.D. Inarrea P. Santos J.C. Lopez-Novoa J.M. Sanchez-Crespo M. Biochem. Biophys. Res. Commun. 1984; 120: 789-796Crossref PubMed Scopus (81) Google Scholar, 55Caramelo C. Fernandez-Gallardo S. Santos J.C. Inarrea P. SanchezCrespo M. Lopez-Novoa J.M. Hernando L. Eur. J. Clin. Invest. 1987; 17: 7-11Crossref PubMed Scopus (69) Google Scholar). In view of the high PAF:AH levels present in blood, this is a surprising result, inferring that this PAF may be protected from PAF:AH.In conclusion, PAF released from embryos and endothelial cells binds to albumin at domain II (amino acids 240–386), protecting it from the hydrolytic effects of PAF:AH in vitro. PAF added to solution does not bind to this site on albumin in vitro. This binding makes extracting and measuring cell-released PAF difficult, but may also act to increase the half-life of PAF released from these cell types. Should such cryptic binding also occur in vivo, it may well influence PAF's half-life and hence its potential to act as a circulating mediator. The impact of such binding by albumin on the kinetics of its recognition by the PAF-receptor in vivorequires investigation. Platelet-activating factor (PAF 1The abbreviations used are: PAF, platelet-activating factor; PAF:AH, PAF-acetylhydrolase; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; BSA, bovine serum albumin; ECCM, endothelial cell-conditioned medium; ECM, embryo-conditioned medium; HTFM, human tubal fluid medium; RIA, radioimmunoassay; ANOVA, analysis of variance. 1The abbreviations used are: PAF, platelet-activating factor; PAF:AH, PAF-acetylhydrolase; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; BSA, bovine serum albumin; ECCM, endothelial cell-conditioned medium; ECM, embryo-conditioned medium; HTFM, human tubal fluid medium; RIA, radioimmunoassay; ANOVA, analysis of variance.; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active ether phospholipid (1Dempoulos C.A. Pinckard R.N. Hanahan D.J. J. Biol. Chem. 1979; 254: 9355-9358Abstract Full Text PDF PubMed Google Scholar, 2Benveniste J. Tence M. Varenne P. Bidault J. Boullet C. Polonsky P. C. R. Seances Acad. Sci. 1979; 289: 1037-1040PubMed Google Scholar). 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. 3Bratton D. Henson P.M. Barnes P.J. Page C.P. Henson P.M. Platelet Activating Factor and Human Disease. Blackwell Scientific Publications, Oxford1989: 23-57Google Scholar for review). PAF synthesis occurred in endothelial cells from diverse vascular beds, including aorta, umbilical vein, and pulmonary artery (4Whatley R.E. Nelson P. Zimmerman G.A. Stevens D.L. Parker C.J. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1988; 264: 6325-6333Abstract Full Text PDF Google Scholar). Several reports" @default.
• W2024587005 created "2016-06-24" @default.
• W2024587005 creator A5035955986 @default.
• W2024587005 creator A5068450777 @default.
• W2024587005 date "1997-07-01" @default.
• W2024587005 modified "2023-09-26" @default.
• W2024587005 title "Studies of the Nature of the Binding by Albumin of Platelet-activating Factor Released from Cells" @default.
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