Platelet-activating factor as an intercellular signal in neutrophil-dependent platelet activation.

The role of platelet-activating factor (PAF) in heterotypic cell to cell interactions in a rabbit neutrophil-platelet mixture model was investigated. Platelets were exposed to each of three chemotactic agonists: PAF, leukotriene B4 (LTB4), or FMLP. Only PAF stimulated aggregation, [3H]serotonin secretion, and cytosolic Ca2+ mobilization in platelets alone. However, platelets were stimulated by LTB4 and FMLP in the presence of neutrophils. This neutrophil-dependent platelet activation was blocked by pretreatment of platelets with PAF receptor antagonists, and was prevented by desensitization of platelets to PAF. Furthermore, the time-course of platelet activation showed a positive correlation with PAF production by neutrophils stimulated with either LTB4 or FMLP. The PAF-mediated neutrophil-platelet interaction was dependent on direct cell to cell contact, as demonstrated by experiments in which the majority of newly formed PAF was neutrophil associated (rather than released). Platelet activation did not occur when the neutrophil-platelet mixture was not stirred, minimizing cell to cell contact, or when platelets were challenged with a cell-free supernatant prepared from neutrophils activated with LTB4 or FMLP. Finally, the neutrophil-platelet interaction was abolished by SC-49992, a peptidomimetic of the fibrinogen binding sequence Arg-Gly-Asp-Phe, indicating a Arg-Gly-Asp-specific recognition mechanism. Our results demonstrate that neutrophil-generated PAF plays a crucial role in neutrophil-dependent platelet activation in this model system. This type of intercellular signaling event may be important in certain inflammatory or thrombotic processes.     

Calmodulin antagonist W-7 inhibits aggregation of human platelets induced by platelet activating factor

Experiments were done to test the hypothesis that aggregation of human platelets induced by platelet activating factor (PAF) may be mediated by calmodulin-dependent processes. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide], a potent calmodulin antagonist, caused dose-dependent inhibition of PAF induced aggregation of human platelets in vitro. The ED50 for W-7 was 51.5 +/- 9.5 microM (mean +/- SEM). This concentration is known to be platelet calmodulin-specific. These data are consistent with the hypothesis.     

Acetyltransferase activity in human platelet microsomes and washed platelets

It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.     

Mechanisms of interaction of a plasmalogenic analog of platelet-activating factor with human platelets.

The interaction of a plasmalogenic analog of platelet-activating factor (1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkenyl-PAF) with human platelets was studied. 1-Alkenyl-PAF induced an increase in intracellular Ca2+ concentration and inhibition of adenylate cyclase at significantly higher concentrations than PAF. 1-Alkenyl-PAF inhibits PAF-induced platelet aggregation but has no effect on ADP- or thrombin-induced aggregation of human platelets. In contrast to PAF, 1-alkenyl-PAF increases [3H]PGE1 binding with human platelets. The properties of 1-alkenyl-PAF as an agonist or antagonist of PAF receptors apparently depend on its concentration in the cell medium. Under physiological conditions 1-alkenyl-PAF might be a natural PAF antagonist acting in the human cardiovascular system.     

Polymorphonuclear leukocyte-platelet interactions: acetylglyceryl ether phosphocholine-induced platelet activation under stimulation with chemotactic peptide

A mixture of rabbit polymorphonuclear leukocytes (PMNs) and platelets at concentrations of 5 X 10(6) PMN and 3.5 X 10(8) platelets/ml Tyrode's solution was stimulated with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). A micromolar concentration of FMLP elicited an immediate weak aggregation, followed by a strong aggregation with a time lag of about 1 min. Microscopic examination showed that the immediate aggregation was due to PMNs and the delayed one was more complex and involved platelets. The delayed aggregation was dependent upon the concentrations of both the PMNs and FMLP. The delayed aggregation was completely blocked by pretreatment of the PMN-platelet mixture with 8 microM CV-3988, a specific receptor antagonist of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), or by the application of platelets desensitized to AGEPC. The time course of AGEPC production by PMNs was well matched to that of the biphasic aggregation response. Furthermore, nordihydroguaiaretic acid inhibited both the AGEPC production by PMNs and the delayed aggregation in a similar dose-dependent manner. These result demonstrate that AGEPC, newly-generated by PMNs under FMLP-stimulation, is of primary importance in platelet aggregation in a PMN-platelet mixed system.     

Production of platelet-activating factor by stimulated human polymorphonuclear leukocytes. Correlation of synthesis with release, functional events, and leukotriene B4 metabolism

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation that is synthesized by several human cell types including polymorphonuclear leukocytes (PMN). We examined the synthesis and release of PAF by stimulated human PMN under several conditions, assayed by the incorporation of [3H]acetate into PAF and by bioassay. PAF synthesis was induced by calcium ionophore A23187 (IoA), opsonized zymosan (OpsZ), and N-formyl-methionyl-leucyl-phenylalanine (FMLP) with the relative order of potency IoA much greater than OpsZ greater than FMLP. A variety of other agonists, including phorbol myristate acetate, an activator of protein kinase C and of PMN functional responses, did not stimulate PAF synthesis. PAF synthesis by PMN in response to IoA, OpsZ, and FMLP was concentration- and time-dependent but release of the phospholipid was not: little PAF (1 to 10%) was released from PMN in suspension regardless of the total amount produced, the agonist, its concentration, the time of incubation, or the concentration of extracellular albumin. This was also the case with functionally altered neutrophils that had been "primed" with cytochalasin B or lipopolysaccharide or that had adhered to surfaces. PAF synthesis was tightly coupled with leukotriene B4 production by adherent PMN as well as by neutrophils in suspension, supporting the hypothesis that the two lipid autacoids may be derived from a common precursor. However, PAF synthesis could be dissociated from aggregation and surface adhesion, indicating that it is not absolutely required for these responses of activated PMN. The total amount of PAF that accumulated, but not the percentage that was released, was altered in adherent PMN compared to cells in suspension. These experiments demonstrate that PAF production and its subsequent processing by human neutrophils are highly regulated events. PAF synthesis is associated with PMN activation, but it is not a requisite for early adhesive responses of neutrophils. Because little of the PAF produced by stimulated PMN is released from the cells, it appears that PAF has an intracellular role in PMN function and/or that it may have novel intercellular effects that do not require release into the fluid phase.     

Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contribution of PAF-releasing factor in serum.

The behavior of platelet-activating factor (PAF) produced in stimulated human polymorphonuclear leukocytes (PMN) was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase (PAF-AH)-deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treated with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellular PAF was similar to that of intracellular PAF produced in stimulated PMN in the presence of PAF-AH-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As these data suggested the occurrence of PAF-releasing factor(s) in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE-Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 240 kDa, which was different from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10(-9) M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s) releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators.     

The involvement of adhesion molecules and lipid mediators in the adhesion of human platelets to eosinophils.

Platelet-leukocyte interactions represent an important determinant of the inflammatory response. Although mechanisms of platelet-neutrophil adhesion were studied extensively, little is known on the mechanisms of platelet-eosinophil interactions. The aim of the present study was to analyze the involvement of adhesion molecules and lipid mediators in platelet-eosinophil adhesion as compared to platelet-neutrophil adhesion. For that purpose human platelets, eosinophils and neutrophils were isolated and platelet-eosinophil and platelet-neutrophil adhesion induced by thrombin (30 mU/ml), LPS (0.01 microg/ml) and fMLP (1 microM) was quantified using the "rosettes" assay. The involvement of adhesion molecules such as selectin P, glycoprotein IIb/IIIa (GPIIb/IIIa) and lipid mediators such as of thromboxane A2 (TXA2), platelet activating factor (PAF) and cysteinyl leukotrienes (cysLTs) were studied using monoclonal antibodies and pharmacological inhibitors, respectively. Thrombin (30 mU/ml), LPS (0.01 microg/ml) and fMLP (1 microM) each of them induced platelet-eosinophil adhesion that was even more pronounced as compared with platelet-neutrophil adhesion induced by the same stimulus. Anti-CD62P antibody (1 microg/ml) and anti-GP IIb/IIIa antibody (abciximab-3 microg/ml) strongly inhibited platelet-eosinophil as well as platelet-neutrophil adhesion. Aspirin inhibited platelet-eosinophil adhesion, while MK 886-a FLAP inhibitor (10 microM), or WEB 2170-a PAF receptor antagonist (100 microM) were less active. On the other hand aspirin, MK 886 and WEB 2170 all three of them inhibited platelet-neutrophil adhesion. In summary, platelets adhered avidly to eosinophils both after activation of platelets by thrombin, eosinophils by fMLP or simultaneous activation of platelets and eosinophils by LPS. Similarly to platelet-neutrophil interaction adhesion of platelets to eosinophils involved not only adhesion molecules (selectin P, GPIIb/IIIa), but also lipid mediators such as TXA2. The involvement of PAF and cysteinyl leukotrienes in platelet-eosinophil adhesion was less pronounced as compared to platelet-neutrophil adhesion.     

Tumor necrosis factor-alpha enhances platelet activation via cathepsin G released from neutrophils

In this paper we show that TNF-alpha enhances platelet activation. Experiments were performed on a human polymorphonuclear neutrophil (PMN)-platelet cooperation system in which PMN, stimulated by FMLP, release cathepsin G (Cat.G), a serine proteinase responsible for the activation of nearby platelets. Pretreatment of the mixed cell suspension with 5 ng/ml TNF-alpha resulted in a strong platelet activation (37.7 +/- 3.2% aggregation; 46.0 +/- 14.4% serotonin release) in response to a weak concentration of FMLP (1.25 x 10(-8) M) inducing by itself only 7.7 +/- 4.0% of aggregation and 3.8 +/- 4.1% of serotonin release (mean +/- SD; n = 10). This effect was concentration dependent (maximum between 5 and 10 ng/ml) and was optimal for a brief preincubation time (5 min). Under these experimental conditions the target of TNF-alpha was PMN, as shown by beta-glucuronidase release. The observed potentiation was modified neither by 0.1 mM acetyl salicylic acid (a cyclo-oxygenase inhibitor) nor by 0.1 mM BN 52021 (a platelet-activating factor antagonist), while such a phenomenon was fully inhibited by 20 micrograms/ml eglin C, a strong and specific inhibitor of the human granulocytic proteinases, elastase and Cat.G. In fact, full inhibition was also observed with 300 nM alpha-1-antichymotrypsin, a specific inhibitor of Cat.G. This clear-cut evidence of Cat.G involvement was substantiated by the enhancement of Cat.G release from FMLP-activated PMN primed with TNF-alpha. These results demonstrate that the priming of PMN by TNF-alpha may modulate the activation of other inflammatory cells, particularly of platelets. It is hypothesized that this phenomenon could contribute to pulmonary pathologies, and more specifically to the adult respiratory distress syndrome, a disease for which PMN, platelet and TNF-alpha involvement has been proposed.     

Synthesis of platelet-activating factor by human blood platelets and leucocytes. Evidence against selective utilization of cellular ether-linked phospholipids

Synthesis of platelet activating factor (PAF) in blood platelet suspensions may be due to leucocyte contamination. We therefore investigated PAF synthesis in human blood platelet suspensions and granulocyte- (PMN)-enriched leucocyte suspensions upon stimulation by thrombin and Ca2+-ionophore A23187, both in the presence and absence of the presumed PAF catabolism inhibitor phenylmethylsulfonyl fluoride (PMSF). PAF synthesis was measured by aggregation of washed rabbit platelets and by [3H]acetate incorporation. In contrast to A23187, thrombin was unable to stimulate PAF synthesis by leucocytes. As thrombin did induce PAF synthesis by platelet suspensions, this was evidently not due to leucocyte contamination. A23187 also induced PAF synthesis by platelets, but this was dependent upon the platelet isolation method and possibly associated activation. The ratio of [3H]acetate incorporation into 1-alkyl- versus 1-acyl-2-acetylglycerophosphocholine upon stimulation of non-PMSF-treated leucocytes and platelets amounted to 12.8 and 1.2, respectively. These values are at least 10-fold higher than the ratio of 1-alkyl versus 1-acyl species in the cellular phosphatidylcholine precursor for PAF. By PMSF pretreatment, the distribution of incorporated [3H]acetate between 1-ether- and 1-ester-linked species became similar to that in the precursor phosphatidylcholines of the respective cell type, due to increased recovery of [3H]acetate in the acyl compounds. Both leucocyte and platelet homogenates rapidly degraded acylacetylglycerophosphocholine to (acetyl)glycerophosphocholine, and this deacylation was inhibited by PMSF pretreatment of the cells. We conclude that upon cell stimulation a phospholipase A2 converts both alkylacylglycerophosphocholine and diacylglycerophosphocholine to the 2-lysoanalogs in a ratio similar to the occurrence of the parent compounds. The acetyltransferase subsequently acetylates both compounds to acylacetylglycerophosphocholine and alkylacetylglycerophosphocholine (PAF), respectively. Deacylation of the 1-ester-linked species, either before or after acetylation, gives the impression of selective utilization of 1-ether-linked species for PAF production. It is only after inhibition of the deacylation by pretreatment of the cells with PMSF that a mainly nondiscriminative use of 1-ether- and 1-ester-linked species by both phospholipase A2 and acetyltransferase becomes evident.     

IL-1-induced adhesion of polymorphonuclear leukocytes to cultured human endothelial cells. Role of platelet-activating factor

In early studies we found that IL-1 stimulated endothelial cells (EC) to produce platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Inasmuch as this phospholipid has a wide range of biologic activities, including polymorphonuclear leukocytes (PMN) aggregation and chemotaxis, we investigated whether EC-associated PAF could contribute to IL-1-induced PMN adhesion to EC. When four selective PAF antagonists were added to IL-1-stimulated EC during the PMN adhesion assay, adhesion was reduced in a concentration-related way. Similarly, pre-treatment of PMN with PAF before the adhesion assay to induce desensitization to this phospholipid reduced PMN adhesion to IL-1-treated EC. However, comparing the time course and the concentration response curve of IL-1-induced EC adhesivity and PAF synthesis, we found that increased EC adhesivity to PMN required a shorter incubation time and lower concentration of IL-1 to become apparent than PAF production. When acetyl-coenzyme A was added to EC cultures at a concentration that raised PAF synthesis by 60%, no significant increase in PMN adhesion was observed. In addition, after 9 to 10 doublings, the EC ability to synthesize PAF decreased by 85 to 90%, whereas IL-1-induced EC adhesivity to PMN was only slightly diminished. When IL-1-alpha and -beta were tested on EC, we observed that both were equally active in promoting PMN adhesion to EC but only the alpha-form was able to stimulate PAF production. When PMN were seeded on IL-1-treated EC, increased amounts of PAF were detected even when EC were fixed; in addition, the inhibitory effect of a PAF antagonist was evident also in these conditions. Overall these results indicate that IL-1-induced PAF production by EC does not significantly contribute to PMN adhesion to them. We hypothesize that the observed inhibitory effect of PAF antagonists and PAF desensitization of PMN might be directed at PAF produced by PMN themselves during adhesion to IL-1-treated EC.     

Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.     

Specific binding sites for platelet activating factor in human lung tissues

Specific and saturable binding of [3H]-labeled 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (PAF) to membrane preparations of human lung tissues is demonstrated. The equilibrium dissociation constant (KD) was determined by Scatchard analysis to be 4.9 (+/- 1.7) X 10(-10)M and the maximal number of binding sites was estimated to be 140 (+/- 37) fmole/mg protein. The binding site is PAF specific and its selectivity toward PAF analogs is very similar to that in rabbit platelets. Two PAF receptor antagonists, kadsurenone and ginkgolide B, previously characterized in platelet systems, also displace the binding of [3H]-PAF to human lung homogenates. These data indicate that human lung tissues contain PAF specific receptors, and binding of PAF to these receptor sites may be the first step to initiate PAF-induced lung pathophysiology.     

Molecular heterogeneity of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

The molecular heterogeneity of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes (PMN) was assessed by both normal and reverse phase high performance liquid chromatography (HPLC). As detected by rabbit platelet stimulation, at least 5 PAF molecules were separated by HPLC. Fast atom bombardment (FAB) mass spectrometry revealed one of these PAFs was acetyl glyceryl ether phosphorylcholine (AGEPC) with a C_16:0 alkyl chain in the $\text{sn}$-1 position. Although the structures of the remaining PAFs are unknown, two of the peaks of PAF activity had the same retention times on reverse phase HPLC as the C₁₅- and C₁₈-saturated alkyl chain AGEPC homologues. These studies indicate that the human PMN produces multiple molecular species of PAF.     

Attenuation of platelet activating factor (PAF)-induced stimulation of rabbit platelet GTPase by phorbol ester, dibutyryl cAMP, and desensitization: concomitant effects on PAF receptor binding characteristics

The GTPase activities of rabbit platelet membrane were stimulated by platelet activating factor (PAF) in a receptor-mediated manner. The activities of the GTPase were investigated in the platelets which had been pretreated with tetradecanoyl phorbol acetate (TPA), dibutyryl cAMP, and PAF. The specific binding of PAF to intact platelet cells was also determined in these treated cells. In platelets which had been pretreated with PAF and then specifically desensitized to PAF, higher concentrations were required for stimulation of the receptor-coupled GTPase. In addition the extent of stimulation of the GTPase by PAF was also decreased. By contrast thrombin stimulation of GTPase activity was unaffected by this process. In platelets pretreated with high levels of dibutyryl cAMP (greater than 4 mM), the specific binding of PAF was reduced to nearly 50% of the control. Although this specific binding apparently was not inhibited by lower concentrations of dibutyryl cAMP (less than 2 mM), PAF could stimulate the receptor-coupled GTPase only to a much lower extent in these treated cells. TPA had virtually no effect on PAF specific binding. However, higher concentrations were needed for stimulation of the GTPase. On the other hand, the extent of PAF stimulation of the GTPase was not altered. Interestingly in the TPA-treated platelet membrane, thrombin stimulated GTPase activity to a higher level than that in untreated platelet membrane. Thus, TPA, dibutyryl cAMP, and desensitization affected the PAF receptor binding and the receptor-coupled GTPase activities in a characteristic fashion. The molecular mechanisms of these effects are discussed.     

Biphasic response to lipopolysaccharide from E. coli in the isolated ventilated blood-perfused rat lung.

We characterised early circulatory and respiratory responses to lipopolysaccharide from E. coli (LPS, serotype 0127:B8) in the isolated, ventilated and perfused rat lung preparation. Lungs were isolated from anaesthetised Wistar rats and perfused with full blood, platelet rich plasma (PRP), platelet poor plasma (PPP) or Krebs-Henseleit solution (KH). LPS (300 microg/ml) injected into the blood-perfused lung induced a characteristic biphasic response consisting of an immediate, transient decrease in respiratory tidal volume and an increase in pulmonary perfusion pressures followed by a delayed decrease in respiratory tidal volume. An immediate respiratory/circulatory response to LPS was of considerable magnitude only in full blood-perfused lung whereas the delayed response was fully expressed irrespective whether blood, PRP, PPP or KH was used for the lung perfusion. Immediate respiratory/circulatory response was inhibited by WEB 2170 (100 microM), a PAF receptor antagonist, and by camonagrel (300 microM), a TXA2 synthase inhibitor, but not by MK 571 (100 microM), a cysteinyl leukotriene receptor antagonist. Delayed respiratory response was inhibited by camonagrel only. In summary, we demonstrated that the immediate coupled respiratory/circulatory response is mediated by blood cell-derived PAF and TXA2 whereas the delayed uncoupled respiratory response is mediated by lung parenchyma-derived TXA2.     

Pro-inflammatory response of alveolar macrophages induced by sulphite: studies with lucigenin-dependent chemiluminescence

Alveolar macrophages (AM) were studied for their capability to release mediators involved in modulation of neutrophil (PMN) functions. Initial responses were induced by sulphite. Supernatants obtained from canine, human and rat AM pre-treated with sulphite in concentrations of 0.1–2 mmol/L enhanced the respiratory burst of canine, human and rat PMN, measured by lucigenin-dependent chemiluminescence (CL). This PMN-stimulating activity exhibited platelet-activating factor (PAF)-like properties, as indicated by desensitization of the PAF receptor, inhibition with PAF antagonists WEB 2086 and CV 3988, and the kinetic CL response like PAF after chloroform extraction of supernatants inhibitable by PAF antagonist CV 3988. These results indicate that AM are triggered by sulphite to release mediators that activate the respiratory burst of PMN, primarily via the PAF receptor. © 1998 John Wiley & Sons, Ltd.     

Involvement of platelet activating factor in immediate heart response to lipopolysaccharide.

Although lipopolysaccharide (LPS) is recognized to induce a biphasic cardiovascular response its mechanism is not fully elucidated. In this study we analysed the involvement of PAF, TXA(2) and cysteinyl leukotrienes (cysLTs) in the acute cardiovascular effects of LPS in the isolated rat heart as well as in delayed phase of LPS response using a surrogate cellular model of the induction of NOS-2 by LPS in mouse macrophages. Perfusion of rat hearts with LPS resulted, in an immediate fall in heart contractility and coronary flow by 2.5 +/- 0.59 ml x min(-1) and 560 +/- 81 mmHg x sec(-1), respectively. This response was fully blocked by platelet activating factor (PAF) antagonist - WEB 2170 and partially inhibited, by inhibitor of cyclooxygenase (indomethacin) or by inhibitor of thromboxane synthase (camonagrel). The inhibition of leukotriene synthesis (BAY x1005) or cysLTs receptors (BAY x7195) was without effect. Administration of stable PAF analog (methylcarbamyl-PAF - MC-PAF) alone, mimicked heart response to LPS. In cultured mouse macrophages, MC-PAF did not induce NOS-2 expression and when given with LPS it slightly potentiated NOS-2 induction by LPS. However, in presence of WEB 2170 NOS-2 induction by LPS was inhibited in a dose-dependent manner. Inhibition of cyclooxygenase and leukotriene pathways had no effect on NOS-2 induced by LPS. These results indicate that PAF and TXA(2) but not cysLTs mediate the instant heart response induced by LPS, while PAF alone mediates a delayed NOS-2 induction by LPS. Accordingly, PAF may constitute the mediator that links acute and delayed phases of LPS-induced cardiovascular response.     

Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli.

Using simultaneous recording of aggregation and chemiluminescence, responses of human polymorphonuclear leukocytes, blood platelets and their mixture were investigated after stimulation by specific as well as non-specific stimuli for each cell. In our experimental settings, aggregation of platelets and PMN leukocytes was increased in the following order of stimuli: PMA相似文献   

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2 alpha

The inhibition of human platelet aggregation produced by PGF2 alpha is not specific for thromboxane A2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF2 alpha (8 microM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 microM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC50 = 0.5 microM), but not PGF2 alpha (28 microM), displaces the specific binding of [3H] 9,11-epoxymethano PGH2 to washed human platelets. PGF2 alpha produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF2 alpha is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF2 alpha. We suggest that the very weak effect of PGF2 alpha on cyclic AMP production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.