Role of Platelet-Actlvatlng-Factor (PAF) on Cellular Responses after Stimulation with Leptospire Lipopolysaccharide

Leptospire lipopolysaccharide (LPS) stimulated the adherence of polymorphonuclear neutrophils (PMNs) to human umbilical vein endothelial cells (HUVEC). Enhanced PMN adherence in response to leptospire LPS can be mediated by platelet-activator-factor (PAF), because a PAF antagonist reduced adherence. Leptospire LPS also induced the adherence platelets or U937. The second experiment involved leptospire LPS elicited platelet aggregation in a PMN-platelet mixture, because leptospire LPS stimulated human PMN but not the human platelets. The platelet response was observed only in the mixture system and was inhibited by a PAF antagonist. PAF could be an important pathogenic factor in human leptospirosis.     

A preferential inhibition by Zn2+ on platelet activating factor- and thrombin-induced serotonin secretion from washed rabbit platelets

Zinc ions at micromolar levels exhibited a significant inhibitory activity toward platelet activating factor (AGEPC)- and thrombin-induced serotonin release from washed rabbit platelets. In the ranges from 25 to 30 microM and 10 to 50 microM, respectively, zinc essentially prevented any serotonin release from 1.25 X 10(8) cells/microliter by 1 X 10(-10) M AGEPC and by 0.2 unit thrombin/ml. This inhibition by zinc ions, in micromolar range, occurred in the presence of 1.0 mM Ca2+. The amount of zinc needed for inhibition was inversely proportional to the amount of AGEPC present and further zinc must be added prior to or at the same time as the AGEPC to be effective. Introduction of zinc ions after the AGEPC essentially abolished the inhibitory properties of this divalent cation. Other cations such as Cu2+, La3+, Cd2+, and Mg2+ were ineffective as inhibitors at concentrations where zinc showed its maximal effects. Under conditions similar to those noted above, aggregation induced by AGEPC was blocked only to the extent of 25% of a control. No inhibitory action by zinc on thrombin-induced aggregation was noted. It is apparent that zinc ions influence a site(s) on the rabbit platelet of considerable importance to the activation (or signaling) process by AGEPC and thrombin in these cells, as expressed by serotonin release. Zinc should provide a suitable probe to explore the mechanism of action of these agonists in their interaction with sensitive cells and to define in more specific biochemical terms the putative receptor for these molecules.     

Metabolism of 1-O-alkyl-2-acetyl-sn-glycerol by washed rabbit platelets: formation of platelet activating factor

A new type of neutral lipid, 1-O-alkyl-2-acetyl-sn-glycerol (AAG), induced a delayed aggregation pattern on interaction with washed rabbit platelets. Although far less potent on a molar basis than platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC, nevertheless this compound caused an aggregation, albeit delayed in time, remarkably similar to that exhibited by AGEPC. In view of the possible formation of AGEPC in this reaction, AAG was incubated with washed rabbit platelets, and a lipid corresponding in chromatographic behavior to AGEPC was isolated and identified as such by a combined gas-liquid chromatography/mass spectrometry technique coupled with selected ion monitoring.     

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.     

Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.     

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.     

Specific binding of phospholipid platelet-activating factor by human platelets

The binding of the phospholipid platelet-activating factor 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) to washed human platelets was more than 80% complete within 2 min, which coincided with the time of initiation of platelet aggregation by AGEPC. Scatchard plot analysis of the binding of [3H]AGEPC to platelets without and with an excess of unlabeled AGEPC revealed two distinct types of binding sites. One platelet site for AGEPC exhibited a high affinity (KD = 37 +/- 13 nM, mean +/- SD), was saturable, and had a low maximal capacity of 1399 +/- 498 (mean +/- SD) molecules of AGEPC/platelet. The other platelet site demonstrated a nearly infinite binding capacity, consistent with nonreceptor uptake of AGEPC into cellular structures. The specificity of the high-affinity binding site for AGEPC was assessed by comparing the capacity of several analogues of AGEPC to inhibit the binding of [3H]AGEPC to platelets and to induce platelet aggregation. An ether linkage in position 1, a short-chain fatty acid in position 2, and a choline moiety in the polar head group proved to be critical both for the binding of [3H]AGEPC to platelets and for the initiation of platelet aggregation. Exposure of platelets to AGEPC for 5 min at 37 degrees C functionally deactivated the exposed platelets to subsequent stimulation by AGEPC, as assessed by diminished aggregation, and concomitantly reduced the specific binding of [3H]AGEPC. Evaluation of the time course of the events of deactivation revealed the loss of an aggregation response to AGEPC after 90 sec at 37 degrees C, despite the retention of up to 50% of the specific binding sites for AGEPC.     

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)     

Effects of a protein kinase C inhibitor, K-252a, on human polymorphonuclear neutrophil responsiveness

We investigated the capacity of K-252a, an inhibitor of rat brain protein kinase C (PKC), to influence polymorphonuclear neutrophil (PMN) PKC and PMN activation with chemically and structurally dissimilar agonists. K-252a inhibited PMN PKC (IC50 = 0.58 microM), and caused a concentration-dependent (0.1-10 microM) inhibition of degranulation elicited with the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), the lipid agonists, 5(S), 12(R)-dihydroxy-5,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and phorbol 12-myristate 13-acetate. Superoxide anion (O2-) production by PMNs exposed to these stimuli as well as sn-1,2-dioctanoylglycerol (diC8) was also suppressed by K-252a. PMN PKC activity was inhibited with concentrations of K-252a which suppressed PMN responsiveness. Therefore, K-252a appears to be a useful probe for examining the role of PKC in the underlying pathway(s) of PMN activation.     

Structural analogs of alkylacetylglycerophosphocholine inhibitory behavior on platelet activation

Several analogs of alkylacetylglycerophosphocholine (AGEPC; platelet-activating factor) were investigated as potential selective inhibitors of AGEPC-induced activation of washed rabbit platelets. Two particular compounds, CV-3988 (rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl-2-thiazolioethyl++ + phosphate) and U66985 (1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium-hexyl ester) emerged as particularly active and effective inhibitors. Aggregation and secretion profiles, as well as the degradation of inositol phospholipids and production of phosphatidic acid, were used as monitors of their inhibitory capabilities. U66985 was the most effective inhibitor, giving an IC50 value of 4.1 +/- 1.5 X 10(-8) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. Phospholipid turnover was blocked completely at this inhibitor concentration. On the other hand, while CV-3988 was an effective inhibitor, a higher concentration was required and a more restricted range of activity was noted with an IC50 value of 5.9 +/- 1.3 X 10(-7) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. While CV-3988 did indeed completely block the turnover of inositol phospholipids and phosphatidic acid formation, these effects were noted at a higher concentration than with U66985. On the basis of data obtained in desensitization experiments with AGEPC and U66985, it appears that each inhibitor occupies the same receptor site as the agonist, AGEPC. These results illustrate the usefulness of these AGEPC analogs in exploring the biochemical characteristics of the interaction of AGEPC with a cell.     

An early transient decrease in phosphatidylinositol 4,5-bisphosphate upon stimulation of rabbit platelets with acetylglycerylether phosphorylcholine (platelet activating factor)

Washed rabbit platelets labeled with [3H]inositol were stimulated with AGEPC (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (5 X 10(-10) M) for various time periods. Within 5 s of the mixing of these platelets with AGEPC, an approximately 25% decrease in the [3H]TPI (phosphatidylinositol 4,5-bisphosphate) was evident; immediately thereafter the radioactivity in TPI increased. These labeled platelets treated with various concentrations of AGEPC for only 5 s indicated a characteristic dose-related decrease in [3H]TPI. Radioactivity in phosphatidylinositol 4-phosphate also appeared to increase after AGEPC-induced stimulation of platelets. Interestingly, within 15 s a 15 to 20% decrease in [3H]PI (phosphatidylinositol) and an increase in [3H]lysoPI was observed. However, [3H]lysoPI could be related only to one-third of the decrease in [3H]PI. LysoGEPC (lyso-1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which is ineffective in the activation of platelets, was unable to cause any changes in the phosphoinositides. The fact that the status of TPI was influenced in a time- and dose-dependent manner and the rapidity with which these changes take place suggest that this inositol phospholipid may be associated closely with the early processes which accompany the interaction of AGEPC with platelets.     

Stimulation of hepatic glycogenolysis by acetylglyceryl ether phosphorylcholine

1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine or acetylglyceryl ether phosphorylcholine (AGEPC) stimulated glycogenolysis in perfused livers from fed rats at concentrations as low as 10(-11) M. At the lower AGEPC concentrations, e.g. 2 X 10(-10) M, a single transient phase of enhanced hepatic glucose output was elicited upon infusion of this agonist. At higher concentrations, e.g. 2 X 10(-8) M, a sharp transient spike of glucose output was observed, followed by a stable elevated steady state rate of glucose output until the AGEPC infusion was terminated. Increased rates of lactate and acetoacetate output and a diminished hepatic oxygen consumption were characteristic of the response of the livers to AGEPC at 2 X 10(-10) M. Neither alpha- nor beta-adrenergic antagonists blocked the glycogenolytic response of AGEPC. Repeated infusion of AGEPC led to homologous desensitization of the response, but the response of the liver to the alpha-adrenergic agonist, phenylephrine, or to glucagon, subsequent to AGEPC stimulation, was unaffected. Increasing the period of perfusion between successive additions of AGEPC, from 7 to 30 min, resulted in an increased glycogenolytic response to this agonist. When the perfusate calcium concentration was reduced from 1.25 to 0.05 mM, the glycogenolytic response to AGEPC was markedly diminished; calcium efflux from the liver following stimulation with AGEPC was not observed. The data presented in this study illustrate a potent agonist effect of AGEPC on the glycogenolytic system in the rat liver.     

Effect of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine on calcium fluxes by human platelet microsomes

Under conditions where optimal concentrations of arachidonic acid, phosphatidic acid, or the calcium ionophore A23187 caused release of 50-95% of calcium from preloaded platelet microsomes, basophil platelet activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) did not cause the release of calcium at concentrations as high as 2 X 10(-5) M. The failure to stimulate calcium release was not due to metabolism or inactivation of AGEPC. These results show that AGEPC is not a calcium ionophore and is unable to directly effect the release of calcium from microsomes by mechanisms other than ionophoric action. The increase in intracellular levels that occurs during AGEPC-induced platelet aggregation must be an indirect effect of the AGEPC.     

Effect of 4-hydroxynonenal on superoxide anion production from primed human neutrophils

HNE (4-hydroxy-2,3-trans-nonenal), an aldehydic product of lipid peroxidation, has been reported to modulate different functional parameters of human and rat neutrophils (PMNs), such as chemiluminescence, migration and some enzymatic activities, thus exerting effects that varied according to the concentration tested. Experiments were done to evaluate the effects of HNE on superoxide anion (O₂^?.) production from human PMNs, isolated from healthy volunteers. After having tested that HNE by itself was not able to activate the cells, comparisons were made between its effects on PMNs, stimulated by either a single stimulus, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or a combination of stimuli, such as FMLP and the neuropeptide substance P (SP; primed PMNs). In the concentration range tested (10^?12–10^?4 M ), HNE inhibited FMLP-evoked O₂^?. production with an IC₅₀ of 11·6 ± 1·5 × 10^?6 M ; at concentrations ≤10^?6 M , HNE enhanced O₂^?. production elicited by FMLP + SP, while higher concentrations were inhibitory. There was a bell-shaped dose–response curve to the enhancing effects of HNE, depending on the incubation time being recorded after only short periods (≤5 min) of the exposure of the cells to HNE; this was not shown by structurally-related aldehydes, such as 2-nonenal and nonanal. These results suggest that low concentrations of HNE may participate in the evolution of the inflammatory process, by contributing to the activation of PMNs. The effects of high concentrations of the aldehyde may represent a mechanism which contributes to the regulation of the extent of the inflammatory response.     

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.     

Platelet activating factor-stimulated formation of inositol triphosphate in platelets and its regulation by various agents including Ca2+, indomethacin, CV-3988, and forskolin

When myo-2-[3H]inositol-labeled rabbit platelets were stimulated with 1 X 10(-9)M sn-3-AGEPC (platelet activating factor) for 5 s, the levels of [3H]inositol monophosphate (IP), [3H]inositol diphosphate (IP2), and [3H]inositol triphosphate (IP3) increased about 1.5-, 3-, and 5-fold, respectively. Formation of these inositol polyphosphates was strikingly independent of extracellular Ca2+. Inactive analogs of sn-3-AGEPC, i.e., lysoGEPC and stereoisomer sn-1-AGEPC, did not cause production of any inositol polyphosphate. Pretreatment of platelets with indomethacin (5 microM) had little effect on this phenomenon. On the other hand, a platelet activating factor antagonist, CV-3988, blocked the AGEPC-stimulated production of radioactive IP, IP2, and IP3. Similarly forskolin, an activator of adenylate cyclase, at 5 microM or above completely abolished AGEPC-induced aggregation, [3H]serotonin secretion, and formation of [3H]inositol polyphosphates. In the light of the emerging role of AGEPC in inflammation, hypotension, and other cardiovascular processes, studies with platelets reported here indicate that forskolin could be a useful tool for manipulating AGEPC responses. It is further concluded that AGEPC-induced formation of inositol polyphosphate is an early response "specific" to AGEPC, mediated via extracellular Ca2+-independent phosphoinositide phosphodiesterase, and could play a role in intracellular Ca2+ mobilization and platelet shape change.     

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.     

Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin

Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation.     

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets. Evidence for thromboxane A2 dependence

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.     

Beta-adrenergic inhibition of AGEPC-stimulated Na+/Ca2+ exchange and AGEPC-induced platelet activation

Recently, AGEPC (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was found to initiate contraction of ileal smooth muscle strips and to enhance Na+/Ca2+ exchange in ileal plasmalemmal vesicles. In the present study, the effects of the smooth muscle relaxant, isoproterenol, on Na+/Ca2+ exchange in rat ileal plasmalemmal vesicles was examined. In this preparation, Na+/Ca2+ exchange was stimulated 131 +/- 8% and 264 +/- 19% by addition of 50 nM and 100 nM AGEPC, respectively. Isoproterenol, a beta-adrenergic agonist, inhibited AGEPC stimulation of Na+/Ca2+ exchange in a dose- and time-dependent manner but had no effect on basal rates of Na+/Ca2+ antiport. At 1 microM, isoproterenol inhibited 86% of the Na+/Ca2+ exchange stimulated by 50 nM AGEPC. Vesicular cAMP levels were increased over 100% following the addition of 1 microM isoproterenol for 30 s. Inhibition of AGEPC-stimulated vesicular Na+/Ca2+ exchange and elevation of vesicular cAMP levels by isoproterenol was prevented by the beta-receptor antagonist propranolol (5 microM), demonstrating that these effects of isoproterenol were mediated by interaction with vesicular beta-adrenergic receptors. Additional studies with washed rabbit platelets demonstrated that isoproterenol inhibited AGEPC-induced aggregation and serotonin release. These effects of isoproterenol were dose- and time-dependent and were antagonized by propranolol. Isoproterenol had no effect on thrombin-induced aggregation and did not change appreciably platelet cAMP levels. Moreover, dibutyryl cAMP could not mimic the effect of isoproterenol to inhibit an AGEPC-induced aggregation. On a molar basis, the inhibitory effects of isoproterenol toward AGEPC action were greater in the ileal preparation than in the platelets. It is suggested that beta-adrenergic agonists may modulate AGEPC-induced ileal Na+/Ca2+ exchange and AGEPC-induced platelet aggregation through cAMP-dependent and-independent mechanisms, respectively.