Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82 X 10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87 X 10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.     

The role of Ca2+ uptake in the response of human platelets to adrenaline and to 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor)

Thrombin induces Ca2+ uptake into both stirred and unstirred human platelets in the presence or absence of acetylsalicylate. This Ca2+ uptake is closely correlated with adenine nucleotide secretion in accord with previous observations [Massini, P. and Lüscher, E.F. (1974) Biochim. Biophys. Acta 372, 109-121] but a low level of secretion is observed in the absence of significant Ca2+ uptake. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkylAcGEPC) also induces Ca2+ uptake into both stirred and unstirred human platelets in the presence and absence of acetylsalicylate. Aggregation and adenine nucleotide secretion induced by 1-O-alkylAcGEPC can be observed in the absence of added fibrinogen, but addition of fibrinogen causes a very marked shift to the left in the dose/response curves for aggregation and Ca2+ uptake induced by 1-O-alkylAcGEPC. In the absence of added fibrinogen a close correlation is observed between Ca2+ uptake and adenine nucleotide secretion induced by 1-O-alkylAcGEPC. In the presence of added fibrinogen significant aggregation can be observed in the absence of detectable Ca2+ uptake. Adrenaline induces Ca2+ uptake only into stirred human platelets in the presence of added fibrinogen. Blockade of secretion, e.g. by addition of acetylsalicylate, also prevents Ca2+ uptake. Addition of adrenaline fails to cause breakdown of phosphatidylinositol or phosphatidylcholine in acetylsalicylate-treated platelets under conditions where such a response is observed on addition of thrombin. We conclude that Ca2+ uptake into human platelets induced by thrombin, 1-O-alkylAcGEPC and adrenaline is closely associated with the secretory response and in some circumstances, e.g. stimulation by thrombin, is clearly a consequence of this latter response. Previous reports of Ca2+ uptake as an initiating event in the response of human platelets to adrenaline [Owen, N.E., Feinberg, H. and Le Breton, G.C. (1981) Am J. Physiol. 239, H483-488] have not been confirmed in this study.     

Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets

The binding profile of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) to washed rabbit platelets was investigated through the use of structural analogs of AGEPC, e.g. U66985, which specifically suppressed AGEPC biological activities on rabbit platelets. This interaction of AGEPC with platelets could be divided into three different components termed A, B, and C. Component A was considered as one of high affinity (Kd = 0.5 X 10(-9) M) and with a low capacity (about 400 sites/platelet). The binding of AGEPC to component A was reversible and was blocked by the inhibitory analogs of AGEPC. This was considered to be the AGEPC receptor site(s). Component B was irreversible in nature and was presumed to be associated with internalization of AGEPC. The latter process was sensitive to the structural inhibitors. Component C was not affected by the inhibitors and probably represented a nonspecific binding to the lipid layer of the membrane. The binding profile of 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine, a biologically inactive and noninhibitory analog of AGEPC, was observed to consist of a single component and was (also) unaffected by the inhibitors. Internalization of AGEPC into rabbit platelets was further examined by the bovine serum albumin extraction method, which was originally developed by Mohandas et al. (Mohandas, N., Wyatt, J., Mel, S. F., Rossi, M. E., and Shohet, S. B. (1982) J. Biol. Chem. 257, 6537-6543). AGEPC was instantly taken up by the cell and internalization into its membrane, where it remained and was not released into cytosol. The internalization of AGEPC was suppressed by pretreating the cells with AGEPC analogs. In platelets desensitized to AGEPC, no down-regulation of the receptor site(s) was observed. The internalization of AGEPC in the desensitized cells was clearly enhanced and this was obvious even in the presence of the AGEPC inhibitor(s). Even in the presence of the inhibitors, effective internalization of AGEPC was also evident in thrombin-treated cells. These results suggested that the internalization of AGEPC was irreversibly enhanced in the platelets which were activated by AGEPC itself as well as by thrombin.     

Characterization of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)-induced protein phosphorylation in rabbit platelets: inhibitory effects of AGEPC analogs

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) induced phosphorylation of two proteins having molecular masses of approximately 20- and 40-kDa in washed rabbit platelets in a concentration- and time-dependent manner. Sequential stimulation with AGEPC did not induce additional protein phosphorylation, supporting the concept of desensitization of the AGEPC receptors responsible for biological activity. AGEPC analogs 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium hexyl ester and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-10'-trimethylammonium decyl ester (U66985 and U66982), containing polar head groups with methylene chain lengths of C6 and C10, did not cause protein phosphorylation, but they did inhibit the AGEPC-induced events. Thus protein phosphorylation is closely associated with the receptor-mediated stimulation of platelets and is a useful indicator of the signaling process initiated through the receptors. Other synthetic analogs of AGEPC such as rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate and 1-(N-n-pentadecylcarbamoyloxy)-2-methoxy-rac-glycero-3-phosphochol ine (CV3988 and U68043) were also shown to be inhibitors of the AGEPC-induced protein phosphorylation. Inhibition by these analogs was specific for AGEPC since there was no observed effect of thrombin, ADP, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and arachidonic acid-induced changes. The extent of inhibition was dependent on the concentration of AGEPC and its analogs and did not change with time after the addition of AGEPC. In platelets incubated with AGEPC analogs before and simultaneously with the addition of AGEPC, protein phosphorylation was prevented; however, addition of AGEPC to platelets shortly before the addition of these analogs showed a high response. In experiments where platelets were previously incubated with AGEPC analogs and washed with buffer containing 0.5% bovine serum albumin, AGEPC-induced protein phosphorylation was recovered to a level of 80%. These observations support the conclusion that AGEPC stimulates platelets through its specific receptor, and that the AGEPC analogs bind to the AGEPC receptor and block that pathway sensitive to AGEPC stimulation but not because of the desensitization of its receptor. On the other hand, in platelets where phosphorylation of the 40-kDa protein was induced by a 2-min preincubation with 3 X 10(-10) M TPA, 5 X 10(-10) M AGEPC-induced serotonin release decreased by 51% compared to a control value.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether) on the arterial wall

The effect of a topical paf-acether superfusion over an injured arterial segment was assessed in the guinea-pig, using an opto-electronic in vivo thrombosis model allowing on-line quantification of small platelet thrombus dynamics. As compared to control, ADP-induced, thromboformation and behaviour, exogenous paf-acether causes a large, dense platelet thrombus, invaded and surrounded by numerous leukocytes, spreading widely over the adjoining, vacuolized, endothelium. Its embolization has to be forced with prostanoids, mepacrine, EDTA, or with a specific paf-acether antagonist (BN 52021). A few minutes after such forced embolization, a new thrombus starts growing at the same site, without renewal of the paf-acether superfusion. This phenomenon of spontaneous reappearance after forced embolization can be followed during several hours. Experiments with labelled paf-acether and the paf-acether antagonist indicate a possible endogenous paf-acether (or paf-acether-like) production triggered by superfusion with exogenous paf-acether.     

Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.     

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF)

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.     

Human platelets stimulated by thrombin produce platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when the degrading enzyme acetyl hydrolase is blocked.

It has been shown [Touqui, Jacquemin & Vargaftig (1983) Thromb. Haemostasis 50, 163; Touqui, Jacquemin & Vargaftig (1983) Biochem. Biophys. Res. Commun. 110, 890-893; Alam, Smith & Melvin (1983) Lipids 18, 534-538; Pieroni & Hanahan (1983) Arch. Biochem. Biophys. 224, 485-493] that rabbit platelets inactivate exogenous PAF (platelet-activating factor, PAF-acether) by a deacetylation-reacylation mechanism. The deacetylation step is catalysed by an acetyl hydrolase sensitive to the serine-hydrolase inhibitor PMSF (phenylmethanesulphonyl fluoride) [Touqui, Jacquemin, Dumarey & Vargaftig (1985) Biochim. Biophys. Acta 833, 111-118]. We report here that human platelets can produce PAF on thrombin stimulation. This production is marginal and transient, reaching a maximum at 10 min and decreasing thereafter. In contrast, 10-12 times more PAF is produced when platelets are treated with PMSF and stimulated with thrombin. Under these conditions, the maximum formation is observed at 30 min and no decline occurs for up to 60 min after stimulation. In addition, these platelets (treated with PMSF and stimulated with thrombin) incorporate exogenous labelled acetate in the 2-position of PAF, probably by an acetyltransferase-dependent mechanism. Production of PAF by human platelets during physiological stimulation can be demonstrated when PAF degradation is suppressed by the acetyl-hydrolase inhibitor PMSF.     

In vivo metabolism of platelet-activating-factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the protozoan Tetrahymena pyriformis

The metabolism of exogenous platelet-activating-factor was studied in the protozoan Tetrahymena pyriformis in vivo. When the cells are exposed to 1.10(-6) M PAF, the molecule is rapidly metabolized to 1-O-alkyl-2-acyl(long chain)-GPC, a major component of the protozoan membranes. The appearance of lyso-PAF from the first minutes even in low levels provides evidence that deacetylation is an intermediate step. After incubation for 30 min, transformation to aminoethyl phosphonolipids is also observed. The fate of PAF in concentrations 1.5.10(-11) M or 1.10(-8) M PAF, was the same. An amount of PAF depending on the external PAF concentration remained intact in the cell even after 1 h incubation. Our results suggest that the easily cultured protozoan can be a useful model for studying PAF's metabolism.     

Relative amounts of 1-O-alkyl- and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in stimulated endothelial cells.

The specific precursor for platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, constitutes 10 per cent of the 1-radyl-2-acyl-sn-glycero-3-phosphocholines in endothelial cells. Stimulation of endothelial cells results in accumulation of PAF and its sn-1-acyl- analog (acylPAF), with acylPAF the predominant product. Mass spectrometry confirmed these relative amounts and confirmed that stimulated endothelial cells accumulate 1-3 ng PAF per million cells. These data suggest that stimulated endothelial cells accumulate both PAF and acylPAF and that the PAF synthetic pathway in endothelial cells is not highly selective for the specific PAF precursor (1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine).     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human fetal membranes and decidua vera

We have previously reported that platelet-activating factor (PAF) is present in human amniotic fluid obtained from women in labor. We have also demonstrated that PAF, lyso-PAF, and alkyl acyl-sn-glycero-3-phosphocholine (AA-GPC) are present in human amnion tissue. In the reported study, we have investigated the enzymes involved in PAF metabolism in amnion tissue and their regulation. A phospholipase A2 activity has been demonstrated in amnion tissue which cleaves alkyl acyl (long-chain) sn-glycero-3-phosphocholine. The enzyme activity is not altered by Ca2+ and is distinctly different from the phospholipase A2 that we have previously characterized in this tissue. Amnion tissue contains acetyltransferase activity which requires Ca2+ and is associated with the microsomal fraction. Acetylhydrolase is also present in the cytosolic fraction of amnion tissue. Acetylhydrolase activity has also been demonstrated in amniotic fluid. The affinities of acetyltransferase (for lyso-PAF) and acetylhydrolase (for PAF) were unaffected by Ca2+. In the presence of Ca2+, however, the specific activity of acetyltransferase was increased four- to fivefold while that of acetylhydrolase was unaffected. Acetyltransferase and acetylhydrolase activities in fetal membranes and decidua were similar and were unchanged with gestational age. The possible role of PAF in the initiation of human parturition is discussed.     

Synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) in exocrine glands and its control by secretagogues

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholines (platelet-activating factor (PAF] stimulate exocytosis in isolated lobules from guinea pig parotid glands or pancreas by an acetylcholine-like mechanism (S?ling, H. D., Eibl, H. J., and Fest, W. (1984) Eur. J. Biochem. 144, 65-72). We show here that both tissues are able to synthetize PAF themselves. Isolated guinea pig parotid gland acini incorporate labeled acetate into the 2-position of PAF. Stimulation with A23187 or carbamoylcholine lead to a significant stimulation of this process. The newly synthetized PAF is partially released into the medium. Addition of lyso-PAF to the incubation medium does not significantly affect the rate of incorporation of labeled acetate into PAF in the absence or presence of carbamoylcholine. Isolated pancreatic lobules are also able to incorporate labeled acetate into PAF, and cholecystokinin and caerulein lead to a strong stimulation of this process. Incorporation of radioactive lyso-PAF into PAF, but not into 1-O-alkyl-2-long chain acyl-sn-glycero-3-phosphocholine was also significantly stimulated by carbamoylcholine in isolated parotid acini. Under these conditions, the time-dependent stimulation of amylase release paralled that of lyso-PAF incorporation into PAF. The same holds for the concentration dependency of the carbachol effect on these two parameters. In isolated pancreatic lobules, caerulein also stimulated the incorporation of lyso-PAF into PAF. Pulse-chase experiments with radioactive lyso-PAF indicate that stimulation of incorporation of radioactive lyso-PAF into PAF represents increased net synthesis of PAF rather than increased PAF-turnover. Using the platelet aggregation test, substantial amounts (0.79 nmol/g) of PAF could be determined in isolated acini from guinea pig parotid glands.     

Effects of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on cardiac function in perfused guinea-pig heart

The direct cardiac action of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) was studied in isolated perfused guinea-pig heart preparations. PAF produced a fall in left ventricular pressure, decreases in the rate of rise of the left ventricular pressure (dp/dt) and coronary flow, but had no effect on heart rate. These results indicate that PAF is a cardiodepressant with inotropic selectivity and this effect on heart is blocked by CV-3988, a specific PAF antagonist.     

Acetylcholine-like effects of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ('platelet-activating factor') and its analogues in exocrine secretory glands

The effect of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (1), 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (2) 1-O-hexadecyl-sn-glycero-3-phosphocholine (3), 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (4) and its enantiomer 3-O-octadecyl-2-O-methyl-sn-glycero-1-phosphocholine (5) on the secretion of amylase from guinea pig isolated parotid gland and exocrine pancreatic lobules was examined. Compounds 1, 2 and 4 led to a significant stimulation of amylase release in both systems, effects being already visible between 10-100 pM. Maximal stimulation with compounds 1 and 2 occurred at 5 nM, with compound 4 at 1 nM. Higher concentrations were less effective and at 0.1 microM stimulation was very low. In contrast, compound 5 showed a continuous increase in activity up to 0.01-0.1 microM without a decrease at at higher concentrations. Compound 3 had no effect. For compound 1, its effects on calcium and lipid metabolism have been analyzed and compared with those of the acetylcholine analogue carbamoylcholine. Compound 1 mimicked in every respect the effects of carbamoylcholine. It stimulated the uptake of 45Ca by isolated parotid gland lobules in a non-ionophoretic way. In isolated pancreatic lobules it enhanced the incorporation of [32P]phosphate into phosphatidic acid, phosphatidylinositol and poly(phosphoinositide), increased the formation of diacylglycerols and triacylglycerol, led to the same two-phasic responses of myo-[3H]inositol-labeled polyphosphoinositides, and initiated a rapid short-lasting formation of free inositol triphosphate. Accordingly, 'platelet activating factor(s)' can affect the function of exocrine glands at low concentrations. The effects observed resemble those produced by acetylcholine and result most likely from the interaction of platelet-activating factor with plasma membrane receptors.     

Kinetics of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine-induced fibrinogen binding to human platelets

Platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) triggers exposure of fibrinogen binding sites on platelets via binding to specific receptors. Comparison of [3H]PAF-acether binding with 125I-fibrinogen binding shows that the rate with which PAF-acether binds to a number of receptors and not the degree of receptor occupancy determines how much fibrinogen binds. At low concentrations of PAF-acether (0.1-1.0 nM) binding site exposure is incomplete and parallels the rate of formation of the PAF-acether-receptor complex. Fibrinogen binding then primarily depends on the concentration of PAF-acether. At a high concentration of PAF-acether (500 nM) binding site exposure is complete within 2-5 min. Fibrinogen binding then depends on the concentration of fibrinogen. Exposure of binding sites in the absence of fibrinogen leads to disappearance of accessible binding sites. At 500 nM PAF-acether, this disappearance is exponential in nature and shows the same characteristics after 5-15 min incubation with fibrinogen as after 60 min. Exposure of binding sites is then complete within 5 min and their disappearance is not disturbed by other processes. At 0.5 nM PAF-acether, the same characteristics are found after 60 min incubation with fibrinogen, but shorter incubation times reveal an ongoing binding site exposure that interferes with the disappearance process. These results demonstrate close coupling between the PAF-acether receptors and fibrinogen binding sites and indicate that the rate of formation of the PAF-acether-receptor complex is a major factor in the regulation of binding site exposure.     

1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine: acetylhydrolase is associated primarily with low density lipoproteins in human blood

The distribution of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine: acetyl hydrolase acetyl hydrolase activity between different types of human plasma lipoproteins was studied. It was found that lipoprotein-depleted plasma is practically devoid of acetyl hydrolase and of almost all acetyl hydrolase activities recovered in the plasma lipoprotein fraction. Among different types of plasma lipoproteins the bulk of acetyl hydrolase is bound to low density lipoproteins; of those not more than 5-10% is associated with high density lipoproteins. Isolated plasma high density lipoproteins do not influence the activity of acetyl hydrolase associated with low density lipoproteins. It is suggested that low and high density lipoprotein acetyl hydrolase may play different functional roles in human plasma.     

Metabolism of platelet-activating factor in human platelets. Transacylase-mediated synthesis of 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine

The present study demonstrates that inactivation of exogenous 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC; platelet-activating factor) by human platelets is mediated by the sequential action of two enzymes, 1) a Ca2+-independent acetylhydrolase recovered in the cytosolic fraction of platelets that deacylates alkylacetyl-GPC forming alkyllyso-GPC and 2) a CoA-independent, N-ethylmaleimide-sensitive transacylase associated with platelet membranes that incorporates a long-chain fatty acid into alkyllyso-GPC to produce alkylacyl-GPC. Separation of platelet phospholipids and subsequent resolution into individual molecular species by high-performance liquid chromatography revealed that the newly formed alkylacyl-GPC was exclusively alkylarachidonoyl-GPC and that the arachidonoyl group for acylation of alkyllyso-GPC was provided by phosphatidylcholine. We conclude that the previously described platelet arachidonoyl transacylase (Kramer, R.M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811) may play an important role in the metabolism of platelet-activating factor.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.