Specific binding sites for 1-O-alkyl-sn-glyceryl-3-phosphorylcholine on intact human blood neutrophils.

Infection, inflammation and allergy are characterized by an infiltration of neutrophils or eosinophils in the tissue and are associated with an increased level of lysophospholipids. In this study it is shown that labeled 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (lyso-paf) binds to intact human blood neutrophils. Unlabeled lyso-paf(500 nM) inhibits the binding of [3H]lyso-paf to neutrophils in the presence of fatty acid-free serum albumin (0.25%). A linear Scatchard plot analysis of the specific [3H]lyso-paf binding to neutrophils revealed a KD value of 9.2 nM with 4,100 lyso-paf binding sites per neutrophil at 4 degrees C. Lyso-paf increased the specific binding of labeled platelet-activating factor ([3H]paf) to neutrophils at 20 degrees C. The increased specific binding of labeled paf to neutrophils could only be demonstrated when sterile cell preparation methods were used. Intact human blood eosinophils did not express specific lyso-paf binding sites. These results suggest that the lyso-paf binding sites on neutrophils have an up-regulatory function for paf which might be involved in neutrophil-mediated disorders.

Specific high affinity binding of platelet activating factor to intact human blood neutrophils and eosinophils.

Neutrophils and eosinophils are involved in various inflammatory reactions such as leukocyte migration, adherence and phagocytosis. A regulation of platelet-activating factor (PAF) receptors in intact human blood neutrophils and eosinophils is clinically important. Intact human blood neutrophils and eosinophils prepared under sterile conditions specifically bound [3H]PAF in the presence of fatty acid-free serum albumin (0.25% BSA). Excess unlabeled PAF (500 nM) or the specific PAF receptor antagonist WEB 2086 (1 microM) inhibited the [3H]PAF binding. PAF receptors on the surface of intact blood neutrophils and eosinophils had high affinity Kd values of 0.55 and 2.3 nM at 4 degrees C. The Bmax values were 200 fmol/2.5 x 10(6) neutrophils and 26 fmol/2.5 x 10(5) eosinophils. PAF receptors on the outer plasma membranes were functionally relevant as high dose PAF displaced WEB 2086 after 3 min preincubation mediating maximal cytosolic [Ca2+]i flux. High doses of PAF or phorbol myristate acetate (PMA) downregulated neutrophils and a low dose PAF decreased the specific [3H]PAF binding to eosinophils determined with WEB 2086 at 20 degrees C. Only neutrophils were significantly upregulated by low dose PAF (5 nM), lyso PAF or low dose PMA (1 nM). Up- and downregulation by PAF itself of neutrophil and eosinophil PAF receptors might explain their desensitization and some clinical controversy concerning the role of PAF in inflammatory and allergic diseases. The latter hypothesis would lead to a novel combination of antagonists against PAF receptors and PAF production.

Non-specific PAF binding to embryonal F9 cells and prostacyclin synthesis in human umbilical vein endothelial cells.

Our data suggest non-specific Paf binding to intact embryonal F9 cells. Non-specific Paf binding might have some physiological relevance when Paf metabolites interfere with prostacyclin synthesis, for example, in umbilical vein endothelial cells.

Human umbilical vein endothelial cells: specific binding of platelet-activating factor and cytosolic calcium flux.

An interaction of the platelet-activating factor (Paf) with endothelial cells was investigated using human umbilical vein endothelial cells. Confluent endothelial cells bound [3H]Paf in the presence of 0.25% fatty acid-free serum albumin after culture in media containing either heat-inactivated foetal calf serum or serum substitute. The Scatchard analysis of the saturated specific [3H]Paf binding showed a Bmax of 2.5 fmol indicating 2800 binding sites per endothelial cell. [3H]Paf binding was partially reversible at 20 degrees and 4 degrees and endothelial cells partially metabolized [3H]Paf at 20 degrees but not at 4 degrees. [3H]Paf binding and Paf-mediated increase of cytosolic free calcium were inhibited by specific Paf receptor antagonists which do not interfere with Paf metabolism. Immortalized umbilical vein endothelial cells bound [3H]Paf specifically after culture in the presence of insulin (20 hr, 0.4 U/mL) with non-specific binding in the absence of insulin. The results show that specific Paf binding mediated calcium flux in human endothelial cells.

Lipoprotein-associated paf (LA-paf) was found in washed human platelets and monocyte/macrophage-like U937 cells.

Beyond cholesterol, inflammatory ether phospholipids such as platelet-activating factor (paf) may play a role in atherogenesis. (1) We detected a paf-like compound ('LA-paf') associated with human serum lipoproteins, mainly in LDL but not with the lipoprotein-poor fraction. (2) LA-paf was also found in washed human platelets, from where it was partially released during platelet aggregation in response to paf (50 nM) or thrombin (1 U). In addition, resident monocyte/macrophage-like U937 cells carried huge amounts of LA-paf (41 ng per 10(7) cells) and metabolized added [3H]paf to a labelled compound co-eluting with the retention time of LA-paf in standard HPLC. (3) Functionally, LA-paf had a comparable potency to synthetic paf, because LA-paf aggregated washed aspirin-treated platelets in a concentration-dependent manner. The specific paf receptor antagonist WEB2086 inhibited the platelet aggregation induced by three distinct LA-paf preparations as compared with synthetic paf with similar inhibitory concentrations (IC50: 35.6 +/- 12.8, 24.0 +/- 4.0, 38.0 +/- 15.8 nM for LA-paf, and 43.6 +/- 6.5 nM for synthetic paf), indicating that LA-paf interacted with paf receptors. (4) However, LA-paf had a distinct retention time using high-pressure liquid chromatography (HPLC) as compared with synthetic paf. LA-paf eluted at 9-15 min and synthetic paf at 21-24 min. In addition, total and non-specific [3H]paf binding to intact washed human platelets was affected differently by the two unlabelled agonists: while LA-paf increased total and non-specific (but not specific) binding in a significant manner (P < 0.002 and P < 0.007) as LDL did (P < 0.006 and P < 0.03), synthetic paf decreased total binding (P < 0.03). Similarly, low-density lipoproteins (LDL) increased significantly the total [3H]paf binding. In contrast, paf did not affect specific [125I]LDL binding to human fibroblasts. Our results show the presence of LA-paf in lipoproteins, washed human platelets and monocyte/macrophage-like cells. As LDL and LA-paf purified from the same LDL particles increased significantly the total [3H]paf binding to intact human platelets, it might modulate platelet adherence to vascular endothelial cells.

Human platelets release a paf-acether: acetylhydrolase similar to that in plasma.

Intact washed human platelets aggregated in response to paf-acether (paf) and did not metabolize [3H]paf at concentrations up to 10 nM. However, when platelets were lysed by exposure to pH 9.5, resulting in 37.5 +/- 2.5% (mean +/- SD, n = 3) lactic dehydrogenase (LDH) release, 20.5 +/- 5.7% of the radioactivity was detected as labeled lyso paf and 5.7 +/- 3.1% as labeled alkylacylglycerophosphocholine. When platelets were aggregated with 0.5 IU/mL thrombin or high concentrations of paf (100 nM), they released a part of their acetylhydrolase without releasing LDH. In supernatants obtained from aggregated platelets, 21 +/- 2% or 10 +/- 2% (n = 3), respectively, of the total platelet acetylhydrolase activity was detected vs. none in supernatants of resting cells. The release of acetylhydrolase was concentration- and time-dependent and paralleled the release of PF 4, a marker for alpha-granules. The acetylhydrolase affinity for paf (Km) measured in sonicates of resting and thrombin-activated platelets was 8.3 +/- 1.5 microM vs. 10.6 +/- 1.5 microM, n = 5, n.s. in a "Mann Whitney" test. The latter Km was slightly but significantly different (P < 0.05, n = 5) from that of the thrombin-released acetylhydrolase (7.9 +/- 1.5 microM) and that of the latter was itself different from plasma acetylhydrolase (5.3 +/- 0.5, P < 0.05, n = 5). Addition of plasma (acid-treated to inactivate acetylhydrolase) decreased the Km value of supernatant acetylhydrolase to 6.1 +/- 1.4 microM. All preparations of acetylhydrolase exhibited similar pH requirements and sensitivity to various inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets.

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellularly. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I2 (PGI2), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI2) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor.

Long time incubation of monocytic U 937 cells with LDL increases specific paf-acether binding and the cellular acetylhydrolase activity.

Besides the well established role of low density lipoproteins (LDL), the phospholipid PAF-acether (paf) seems to be involved in atherogenesis. The effect of LDL (10 micrograms/ml for 24 h, n = 3) on paf binding characteristics of monocyte/macrophage-like U 937 cells was investigated using the radioligand [3H]paf, unlabeled paf and the paf receptor antagonist WEB 2086. The specific [3H]paf binding significantly increased at 1.4 nM (P less than 0.02) and 2.8 nM (P less than 0.01) added [3H]paf with an increased number of paf binding sites in the Scatchard plot analysis of the data. Specific paf binding was functionally active since paf mediated a cellular [Ca2+]i rise. The protein kinase C (PKC) activator PMA (1 nM, 37 degrees C) expressed specific [3H]paf binding already after a 15-min incubation period, indicating a PKC activation as the decisive step of paf receptor expression. LDL also stimulated the paf degrading cellular acetylhydrolase significantly by increasing both Km (9.4 +/- 1.9 vs. 2.0 +/- 0.5 microM, P less than 0.02) and vmax (0.5 +/- 0.2 vs. 0.2 +/- 0.0 nmol/min per mg cell protein, P less than 0.02). The data demonstrate that LDL increases the number of paf receptors on monocyte/macrophage-like U 937 cells and interferes with the dynamics and/or synthesis of the cellular acetyl hydrolase. These effects could be of importance in the pathogenesis of atherosclerosis.

1,25-(OH)2 vitamin D-3 enhances Paf-stimulated calcium mobilization in U937 cells by expression of specific Paf binding sites.

The effect of 1,25-(OH)2 vitamin D-3 (10 nM, 72 h) on cytosolic free Ca2+ concentration ([Ca2+]i) in U937 cells before and after stimulation with Paf, LTD4 and ADP was investigated. 1,25-(OH)2 vitamin D-3 increased basal [Ca2+]i from 98 +/- 1 nM to 121 +/- 5 nM (P less than 0.01) and the Paf (10 nM) stimulated increase in [Ca2+]i from 143 +/- 15 to 406 +/- 44 nM (P less than 0.01). These vitamin D-3 effects were time-related and occurred after 24 h (basal [Ca2+]i) and 12 h (Paf stimulated Ca2(+)-mobilization) but not after 3 h. In comparison, vitamin D-3 failed to modulate Ca2(+)-mobilization in response to ADP (1-40 microM) and increased it only in response to low leukotriene D4 concentrations (0.1-1 nM). The total binding of [3H]Paf (2.8 nM) was not significantly different in untreated vs. vitamin D-3-treated cells. However, the Paf receptor antagonist Web 2086 (1 microM) inhibited [3H]Paf binding only in vitamin D-3-treated cells. The specific binding reached a plateau of 28 +/- 3 fmol per 2.5.10(6) cells between 1.4 and 2.8 nM [3H]Paf. The Paf receptor antagonist Web 2086 (1-1000 nM) also inhibited the Paf-mediated Ca2(+)-mobilization in vitamin D-3-treated cells (IC50 = 191 +/- 55 M). These data suggest that the enhanced Ca2(+)-mobilization in vitamin D-3-treated U937 cells in response to Paf is mediated by an expression of putative Paf receptors.

Interaction of the Paf antagonist WEB 2086 and its hetrazepine analogues with human platelets and endothelial cells.

1. Intact platelets and confluent human umbilical vein endothelial cells bound [3H]-Paf-acether (platelet activating factor, [3H]-Paf) at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA). 2. [3H]-Paf binding to platelets was inhibited in a concentration-dependent manner by WEB 2086. An excess of WEB 2086 indicated the presence of specific, saturable Paf binding which reached a maximum of 28.3 +/- 3.7 fmol [3H]-Paf per 5 x 10(7) platelets. In platelets, different hetrazepines (WEB 2098, 2105, but not 2118) also inhibited [3H]-Paf binding in a concentration-dependent manner. 3. WEB 2086 partially displaced platelet-bound [3H]-Paf in a concentration-dependent manner reaching a plateau at 400 nM WEB 2086. No further displacement was observed when WEB 2086 and an excess of unlabelled Paf were added together. 4. The hetrazepines inhibited platelet aggregation. Platelet aggregation IC50 values correlated well with the IC50 values of the hetrazepines against [3H]-Paf binding (r2 = 0.99). WEB 2086 shifted the Paf dose-response curve rightwards in a parallel manner. Tested against platelet aggregation the pA2 obtained for WEB 2086 was 7.9. 5. WEB 2086 inhibited [3H]-Paf binding to endothelial cells in a concentration-dependent manner. WEB 2086 also inhibited the Paf-mediated cytosolic calcium increase in endothelial cells with an IC50 value of 23.1 +/- 10.4 nM as compared with an IC50 of 21.6 +/- 10.4 nM WEB 2086 for platelet aggregation. 6. These results demonstrate an inhibition of [3H]-Paf binding to platelets and endothelial cells by different hetrazepines, most probably at the Paf receptor level.

Günther vena caval filter: results of long-term follow-up.

A Günther vena caval filter was implanted in the inferior vena cava in 59 patients to prevent pulmonary embolism. This newly available device, which can be inserted percutaneously via a 10-French introduction system, has three filtering planes. No complications occurred at the puncture site. Follow-up included clinical examinations (54 patients), plain radiographs (50 patients), and CT scans (41 patients); these examinations were performed up to 21 months after implantation. Caudal migration of the filter occurred in 35 (70%) of the 50 patients who had radiographs, but no cranial or oblique movement occurred. Occlusion of the filter was noted in three (7%) of 41 patients who had CT examinations. Thromboemboli were seen inside the filter in 16 (39%) of the 41 patients who had CT scans. Recurrent pulmonary embolism was not observed after filter implantation. The Günther vena caval filter seems to be a satisfactory device for preventing pulmonary embolism.

Comparison of three paf-acether receptor antagonist ginkgolides.

The aggregation of washed human platelets with paf-acether (paf) and [3H]paf binding were inhibited in a concentration- and time-dependent manner by three chemically defined ginkgolides (BN 52020, BN 52021, BN 52022) and their mixture, BN 52063 (molar ratio: 2:2:1). The IC50 values for aggregation correlated well with the IC50 values for [3H]paf binding (R-square 0.971). BN 52021, BN 52020 and BN 52063 (6 microM), as well as unlabelled paf (50 nM), displaced platelet-bound [3H]paf. Specific binding (total minus non-specific binding) in the presence of BN 52020 and BN 52063 was saturated but that observed in the presence of BN 52022 was not. However all ginkgolides shifted the dose-dependent paf-induced platelet aggregation curve rightwards. BN 52063 failed to modulate paf metabolism either in plasma or in the presence of platelets. The present results suggest that the ginkgolide mixture, BN 52063, acts at the paf binding level and the close correlation between ginkgolide effects on aggregation vs. paf binding illustrates the functional relevance of the putative paf platelet receptor.

Preformed PAF-acether and lyso PAF-acether are bound to blood lipoproteins.

PAF-acether (PAF) is a newly formed mediator not normally present in circulating blood. A compound exhibiting all of its biological characteristics but coeluting with phosphatidylcholine (PC) in high-pressure liquid chromatography (HPLC) was unveiled ('peak X') in normal human plasma. A second HPLC run of peak X HPLC fractions revealed the presence of PAF itself with concomitant disappearance of peak X. Beside PAF, immunoreactive apolipoproteins A-I and E were found in peak X. Also lipoproteins (Ls) purified using either ultracentrifugation or immunoaffinity chromatography yielded peak X and, in a second HPLC run, authentic PAF. L-free plasma was devoid of peak X. Finally, after preincubation with plasma, labeled PAF was found associated with Ls. Thus in human blood preformed PAF is bound in high amounts to Ls, a result of interest given the role of Ls and platelets in vascular diseases and the present knowledge on PAF biosynthesis.

Lack of correlation between cytotoxicity of agonists and antagonists of platelet activating factor [paf-acether] in neoplastic cells and modulation of [3H]-paf-acether binding to platelets from humans in vitro.

The 3 ether-lipids ET-18-OCH3, SRI 63-154 and paf-acether, the TLP BM 41.440, the ester-linked 2-LPC and CV-3988, were tested for cytostatic/antiproliferative [3H]-thymidine uptake) and cytotoxic (trypan blue dye exclusion, HTCA) activity in 11 neoplastic human cell lines (U 698-M, Nall-1, Su-DHL-4, RPMI 8226, K 562-4, Li-A, HTB-47, HTB-38, CCL218, 85 HG-59, 85 HG-63) and 1 ALL in vitro. 2-LPC and paf-acether showed either no, or only minor, CV-3988 varying activity. There were no significant differences in the activity of ET-18-OCH3, SRI63-154 and BM 41.440, which showed IC50- and LC50-values of less than or equal to 10 micrograms/ml after incubation periods greater than or equal to 48 hours with or during continuous exposure to the cells. The latter three compounds were then tested for interaction with [3H]-paf-acether binding to intact human platelets: ET-18-OCH3 and SRI63-154 reduced [3H]-paf-acether binding in a time-dependent manner. BM 41.440 did not show this interaction. Thus, since the in vitro cytotoxicity of these lipids did not correlate with their modulation of [3H]-paf-acether binding to human platelets, it was concluded that cytotoxicity of ether-lipids is not mediated by specific paf-acether binding sites similar to those present on human platelets. This finding is important for the future design of antineoplastic lipids.

BN 52021 displaces [3H]paf-acether from, and inhibits its binding to intact human platelets.

Intact platelets incubated at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA) bound [3H]paf-acether in a concentration-dependent (0-6.5 nM) manner. Specific [3H]paf-acether binding in the presence of unlabelled paf-acether or BN 52021, a chemically defined ginkgolide, reached a plateau of 14.5 +/- 5 or 17.5 +/- 7.0 fmol at concentrations higher than 0.65 nM [3H]paf-acether. Unlabelled paf-acether or BN 52021 inhibited and displaced [3H]paf-acether binding in a concentration- and time-dependent manner reaching a plateau at 5 nM or 5 microM. Unlabelled paf-acether inhibited [3H]paf-acether binding (75.1 +/- 8%) more strongly if labelled and unlabelled ligands were incubated together than after 15 min preincubation with [3H]paf-acether alone (32.2 +/- 8.6%). The enantiomer of paf-acether (50 nM) failed to displace platelet-bound [3H]paf-acether and the possibility of degradation of [3H]paf-acether by platelets or BN 52021 was excluded under binding conditions. The results demonstrate the presence of specific binding sites for paf-acether in human platelets and the direct competition by BN 52021 for these sites. Serum albumin inhibited total and enhanced specific [3H]paf-acether binding. Radiolabelled albumin itself did not bind to human platelets at 20 degrees C and preincubation of platelets with antibodies to human serum albumin did not inhibit specific paf-acether binding but increased total [3H]paf-acether binding slightly. Serum albumin appears as a necessary phospholipid carrier for specific [3H]paf-acether binding.

Specific inhibition of PAF-acether-induced platelet activation by BN 52021 and comparison with the PAF-acether inhibitors kadsurenone and CV 3988.

BN 52021 is a chemically defined substance extracted from Ginkgo biloba leaves. Its inhibitory potency was tested on washed human platelets prepared so as to render them specifically sensitivity either to adenosine 5'-diphosphate (ADP), arachidonic acid (AA) or PAF-acether. Its activity and specificity were compared with those of two other reported inhibitors of PAF-acether effects: Kadsurenone and CV 3988. PAF-acether-induced aggregation of washed human platelets was concentration dependently inhibited by BN 52021 (IC50: 2.22 +/- 0.79 microM against 7.5 nM PAF-acether (n = 3)). Under the same experimental conditions the aggregation triggered by ADP was not modified and that induced by AA was marginally affected. The PAF-acether EC50 in platelet-rich plasma was increased 5- and 46-fold with 1 microM and 5 microM of BN 52021 respectively. This strongly suggested that the mechanism of action of BN 52021 is of the competitive type. Analysis of [3H]PAF-acether binding showed that BN 52021 as well as unlabelled PAF-acether prevented [3H]PAF-acether binding to intact washed platelets. In washed human platelets Kadsurenone affected only PAF-acether-induced aggregation (IC50: 0.8 +/- 0.4 microM (n = 3)), whereas CV 3988 inhibited the aggregation induced by ADP, AA and PAF-acether (IC50 were 10.2 +/- 2.3 microM; 2.2 +/- 0.1 microM; 1.0 +/- 0.1 microM respectively (n = 3). In contrast, up to 30 microM, CV 3988 was a specific antagonist of PAF-acether-induced platelet aggregation in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Unsaturated platelet-activating factor: influence on aggregation, serotonin release and thromboxane synthesis of human thrombocytes.

Unsaturated platelet-activating factor (paf-acether) aggregated thrombocytes of healthy male volunteers like saturated paf-acether. Unsaturated paf-acether released serotonin in the presence of imipramine. Within one minute the release increased depending on the concentrations of unsaturated paf-acether up to 45% of the serotonin. Human thrombocytes synthesized only a small amount of thromboxane B2 (TXB2) after aggregation induced by unsaturated paf-acether. Unsaturated paf-acether prevented the binding of radiolabelled saturated paf-acether to intact washed thrombocytes in the same extent as saturated paf-acether.

Unsaturated 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (unsaturated platelet-activating factor): aggregation of human platelets after incubation with indomethacin, creatinephosphate/creatinephosphokinase, xylocain and hirudine, and serotonin release after incubation with indomethacin.

Unsaturated platelet-activating factor (PAF) aggregates thrombocytes of healthy female volunteers and releases within 1 min up to 30.95% of the platelet serotonin. Indomethacin does not inhibit the aggregation but reduces the release of serotonin induced by unsaturated PAF in citrated platelet-rich plasma (PRP). Creatinephosphate combined with creatinephosphokinase (CP/CPK) inhibits the second phase, whereas xylocain inhibits the first and second phase of aggregation induced by unsaturated PAF. Hirudine shows no influence on the aggregation induced by unsaturated PAF.

1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines: influence on aggregation and [3H]serotonin release of human thrombocytes.

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) aggregate human thrombocytes in a concentration dependent fashion. After a short lag-phase, maximum aggregation is reached within 2 min. PAF releases serotonin from human thrombocytes within 1 min. Indomethacin and creatine phosphate (CP)/creatine phosphokinase (CPK) are able to inhibit the second phase of the aggregation by PAF, while xylocain reduces both the first and second phase of aggregation of human thrombocytes. Hirudine neither influences the first nor the second phase of aggregation by PAF.