A new method of purification and sensitive bioassay of platelet- activating factor (PAF) in human whole blood

doi:10.1016/0024-3205(94)00701-2

Life Sciences

Volume 54, Issue 6, 1994, Pages 429-437

https://doi.org/10.1016/0024-3205(94)00701-2 Get rights and content

Abstract

There is no satisfactory assay procedure of PAF in human whole blood in terms of sensitivity, reproducibility and simplicity. This is due to coexisting lipids from plasma and cellular membranes which inhibit measurement of PAF in various assay procedures, including bioassay. In the present study, an attempt was made to eliminate these interfering lipid inhibitors from blood samples. Lipids in human whole blood were extracted according to the method of Bligh & Dyer and the organic layer was dried under a stream of nitrogen. Then, the dried organic layer was dissolved in diethyl-ether and the solution was kept at -20°C which was then centrifuged. The resulting supernatant was then applied to an anion-exchange column and the PAF fraction was obtained by step-wise gradient elution. The fraction was further purified by normal phase HPLC. Then PAF in the final sample was determined by sensitive bioassay using rabbit platelets containing fibrinogen and epinephrine. The recovery rate of PAF throughout this procedure was constant and satisfactory (37.4±9.7%), which was confirmed using [³H]-PAF. The lower limit of the present assay was estimated to be 5pg in 1 ml of blood and it was sensitive enough to detect PAF in blood samples from healthy volunteers and patients with sepsis or liver cirrhosis. Furthermore, attempts were made to compare the sensitivity and the recovery of our method with these of a commercially available radioimmunoassay (RIA) kit for PAF. However, it was not possible to detect any amount of authentic PAF added to whole blood.

References (23)

K. Yamada et al.
J. Chromatogr.
(1988)
S.J. Cooney et al.
J. Immunol. Methods.
(1992)
J. Sugatani et al.
Life Sci.
(1990)
T. Kawasaki et al.
Thromb. Res.
(1984)
J. Casals-Stenzel
Eur. J. Pharmacol.
(1987)
C. Caramelo et al.
Biochem. Biophys. Res. Commun.
(1984)
J.C. Ludwig et al.
Arch. Biochem. Biophys.
(1984)
F. Snider
Am. J. Physiol.
(1990)
C.S. Ramesha et al.
Biomed. Environ. Mass Spectrom.
(1986)
D.J. Hanahan et al.
Methods Biochem Anal.
(1985)

M.J. Bossant et al.

Methods Enzymol.

(1990)

Cited by (14)

Platelets in the immune response: Revisiting platelet-activating factor in anaphylaxis
2015, Journal of Allergy and Clinical Immunology
Citation Excerpt :
In human sepsis studies findings have been conflicting.76 Some studies have found increased serum PAF levels in septic patients,79,80 but others have found no significant increase compared with levels in healthy control subjects.81,82 Plasma PAF-AH activity has also been found to be decreased in some patients with sepsis, raising the possibility of a pathogenic imbalance between increased PAF generation and decreased degradation.46
Anaphylaxis is an acute, severe, life-threatening multisystem allergic reaction resulting from the sudden systemic release of biochemical mediators and chemotactic substances. Release of both preformed granule-associated mediators and newly generated lipid-derived mediators contributes to the amplification and prolongation of anaphylaxis. Platelet-activating factor (PAF) is a potent phospholipid-derived mediator the central role of which has been well established in experimental models of both immune-mediated and non–immune mediated anaphylaxis. It is produced and secreted by several types of cells, including mast cells, monocytes, tissue macrophages, platelets, eosinophils, endothelial cells, and neutrophils. PAF is implicated in platelet aggregation and activation through release of vasoactive amines in the inflammatory response, resulting in increased vascular permeability, circulatory collapse, decreased cardiac output, and various other biological effects. PAF is rapidly hydrolyzed and degraded to an inactive metabolite, lysoPAF, by the enzyme PAF acetylhydrolase, the activity of which has shown to correlate inversely with PAF levels and predispose to severe anaphylaxis. In addition to its role in anaphylaxis, PAF has also been implicated as a mediator in both allergic and nonallergic inflammatory diseases, including allergic rhinitis, sepsis, atherosclerotic disease, and malignancy, in which PAF signaling has an established role. The therapeutic role of PAF antagonism has been investigated for several diseases, with variable results thus far. Further investigation of its role in pathology and therapeutic modulation is highly anticipated because of the pressing need for more selective and targeted therapy for the management of severe anaphylaxis.
Kupffer cells are a major source of increased platelet activating factor in the CCl<inf>4</inf>-induced cirrhotic rat liver
2003, Journal of Hepatology
Background/Aims: Endothelin-1 (ET-1) stimulates the synthesis of platelet-activating factor (PAF) by Kupffer cells in vitro. Hepatic concentrations of both ET-1 (a potent vasoconstrictor) and PAF (a mediator of hepatic vasoconstriction and the cirrhotic hyperdynamic state) increase in cirrhosis. The aim of this study was to determine if the responsiveness of Kupffer cells to produce PAF upon ET-1 challenge is modified by cirrhosis.
Methods: Kupffer cells, isolated from the livers of control and CCl₄-induced cirrhotic rats, were placed in serum-free medium after overnight culture. PAF and ET-1 receptors, ET-1-induced PAF synthesis, and PAF- and ET-1-induced prostaglandin E₂ (PGE₂) synthesis were determined 24 h later.
Results: Both basal and ET-1-stimulated PAF synthesis was increased in cirrhotic Kupffer cells as indicated by increased cell-associated and released PAF. Cirrhotic Kupffer cells also had elevated densities of functional receptors for both PAF and ET-1 (exclusively ET_B), as measured by ligand binding, mRNA expression of the respective receptors, and ligand-stimulated PGE₂ synthesis.
Conclusions: Cirrhosis sensitizes Kupffer cells to both ET-1 and PAF by elevating their respective receptor levels. Since both mediators individually cause portal hypertension, an increase in ET-1-stimulated PAF synthesis in Kupffer cells will exacerbate the hepatic and extrahepatic complications of cirrhosis.
The release mechanism of platelet-activating factor during shear-stress induced platelet aggregation
1997, Biochemical and Biophysical Research Communications
We previously reported that 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF), released from activated platelets stimulated with thrombin plus collagen, is associated with platelet microparticles. In the present study, we found that PAF is concentrated and associated with microparticles released from high shear stress-induced activated platelets. The total amount of PAF released from 3×10⁸platelets under high shear stress (108 dyne/cm²) was 3.2±0.6×10⁻¹⁵mol (n=5, mean± S.D.). Eighty percent of the PAF released from the platelets was recovered in the microparticle fraction after ultracentrifugation in the presence of albumin. Under high shear stress, PAF was not released from platelets within 3 minutes, although microparticles were released. In conclusion, microparticles released from activated platelets in the rheological condition of high shear stress are major carriers of PAF.
Platelet microparticles: A carrier of platelet-activating factor?
1996, Biochemical and Biophysical Research Communications
Platelet-activating factor (PAF) has been considered to be released in a soluble form from activated platelets. Also a considerable number of microparticles were released from stimulated platelets. To evaluate the possibility that microparticles contain PAF, the amount of PAF in whole activated platelets, microparticles and the suspending medium was determined by means of bioassay. The total amount of PAF released from 3 × 10⁸platelets activated with thrombin (0.5 U/ml) plus collegan (10 μg/ml) for 10 minutes was 4.6 ± 1.5 × 10⁻¹²mol (n = 8, mean ± S.D.). Eighty-seven percent of the PAF released from platelets was associated with microparticles. PAF released in microparticles is suspectible to plasma acetylhydrolase and may be localized on the outer surface of microparticles. This is the first report implicating the role of microparticles as a novel carrier of mediators formed in the cells.
Feed-forward reciprocal activation of PAFR and STAT3 regulates epithelial-mesenchymal transition in non-small cell lung cancer
2015, Cancer Research
Platelet-activating factor receptor-mediated PI3K/AKT activation contributes to the malignant development of esophageal squamous cell carcinoma
2015, Oncogene

View all citing articles on Scopus

View full text

A new method of purification and sensitive bioassay of platelet- activating factor (PAF) in human whole blood

Abstract

J. Chromatogr.

J. Immunol. Methods.

Life Sci.

Thromb. Res.

Eur. J. Pharmacol.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Am. J. Physiol.

Biomed. Environ. Mass Spectrom.

Methods Biochem Anal.

Methods Enzymol.