Biomedical subjects

D J Hanahan

Publications and source records attributed to D J Hanahan.

At least 37 records · Page 2Linked to original sources

PubMed1990

Regulation of platelet-activating factor receptor and PAF receptor-mediated arachidonic acid release by protein kinase C activation in rat Kupffer cells.

Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C activator, caused down-regulation of receptors for platelet-activating factor (AGEPC) on the plasma membrane of rat Kupffer cells (40-50% reduction) but had a relatively minor effect on the binding affinity of the receptors for AGEPC (Kd = 0.30 nM vs 0.56 nM) when incubated with the cells for a short period of time (30-60 min). As a consequence, the AGEPC receptor-mediated arachidonic acid release was attenuated. The PMA-induced down-regulation of AGEPC receptors was concentration-dependent, specific, and transient (the maximal effect was observed at about 1 h and the level of specific [3H]AGEPC binding gradually returned to the control level within 8.5 h and even higher than the control level at 24 h after addition of PMA). Upon removing PMA from the culture medium, more than half of the lost receptors were replaced within 1 h at 37 degrees C and the recovery process appeared to be independent of protein synthesis. The ability of PMA to down-regulate the AGEPC receptors was lost in cells "down-regulated" for protein kinase C, suggesting that the receptor-regulatory effect of PMA is protein kinase C-dependent. Protein kinase C appeared to be involved in the AGEPC-induced arachidonic acid release since 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride, a protein kinase C inhibitor, attenuated the stimulatory effect of AGEPC in this system. In addition, AGEPC-induced [3H]arachidonic acid release was inhibited significantly in cells down-regulated for protein kinase C. The present study thus demonstrates that protein kinase C has dual actions in the regulation of AGEPC-mediated events, i.e., a positive forward action, regulating AGEPC-stimulated arachidonic acid release, and a negative action, which inactivates or down-regulates AGEPC receptors.

Animals↗

PubMed1990

Isolation of platelet-activating factor and purification by thin-layer chromatography.

Isolation and purification of platelet-activating factor can be accomplished with reasonable ease and effectiveness given the precautions and suggestions outlined above. These experimental approaches represent a composite of those practiced in the author's laboratory combined with selected observations reported in the literature.

Chromatography, Thin Layer↗

PubMed1990

Novel route to preparation of high purity lysoplasmenylethanolamine.

A rapid and simple method has been developed for the preparation of highly purified lysoplasmenylethanolamine. The starting material, a phosphatidylethanolamine (PE) sample that contained a mixture of the 1, 2 diacyl- and 1-O-alkenyl-2-acyl forms was subjected to mild alkaline methanolysis for 20 min at room temperature. Addition of chloroform and water with vigorous mixing, but without acidification at this point, led to a preferential retention of the lysoplasmenylethanolamine in the alkaline aqueous phase and complete separation of the methyl esters into the chloroform phase. Neutralization of the alkaline phase with dilute acetic acid, followed by addition of chloroform, allowed recovery of the lysoplasmenylethanolamine in the chloroform phase in very high yields (75-80% based on vinyl ether content of starting material). On the other hand, a preparation of cholineglycerophospholipids enriched in plasmenylcholine, treated in exactly the same manner, gave a lysoplasmenylcholine that was not retained in the alkaline phase, but partitioned primarily into the chloroform-rich phase together with the methyl esters. Characterization of the purified lysoplasmenylethanolamine was achieved by thin-layer chromatography and compositional analysis. In addition, fast atom bombardment mass spectral analysis of the intact lysoplasmenylethanolamine together with gas chromatography-mass spectrometry of the dimethyl acetals derived from the 1-O-alkenyl chains allowed further proof of the structure and an assessment of the purity of this compound.

Chromatography, Thin Layer↗

PubMed1989

Regulation of platelet-activating factor receptors in rat Kupffer cells.

Ligand binding studies indicate that 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) down-regulates its own receptors on the plasma membrane of isolated rat Kupffer cells but has no significant effect on the binding affinity of the receptor for AGEPC. Exposure of isolated rat Kupffer cells to 10(-8) and 10(-6) M AGEPC resulted in a rapid, time-dependent reduction in the number of cell surface AGEPC receptors to a new steady state concentration (54.1 +/- 5.0% and 38.6 +/- 5.4% of control, respectively). During the observation period (6 h), the half-time of surface AGEPC receptors was about 60 and 45 min in the presence of 10(-8) and 10(-6) M AGEPC, respectively. Both the rate of loss and the maximal loss of the receptors were dependent upon the AGEPC concentration. With receptor synthesis inhibited by cycloheximide in the absence of AGEPC, the half-time of the surface AGEPC receptor was about 4 h, suggesting that AGEPC receptors are not recycled and that the loss of AGEPC receptors from the plasma membrane is accelerated by AGEPC binding. When incubated with Kupffer cells at 37 degrees C for 3 h, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1.0 microM), an inactive metabolite of AGEPC, did not cause the loss of AGEPC receptors. Under the same conditions, AGEPC antagonists such as BN52021 (2 x 10(-5) M) or U66985 (2 x 10(-5) M) alone had no effect (97.0 +/- 3.9% of control for BN52021) or only a relatively slight effect (78.4 +/- 1.8% for U66985) on the number of surface AGEPC receptors. However, AGEPC antagonists inhibited the AGEPC-induced down-regulation of AGEPC receptors in a concentration-dependent manner, suggesting that the AGEPC-induced down-regulation of AGEPC receptors is a receptor-mediated process. The AGEPC-mediated decrease in receptor number on rat Kupffer cells is reversible. Upon removing AGEPC from the culture medium, about 67% of the lost receptors were replaced within 2 h. Cycloheximide, an inhibitor of protein synthesis, prevented the restoration of the AGEPC receptors. Similar results were obtained when Kupffer cells were incubated with Pronase followed by removing Pronase and reincubating the cells with or without cycloheximide. These observations suggest that the restored AGEPC receptor is newly synthesized rather than recycled. The present study demonstrates that under non-stimulatory (i.e. in the absence of AGEPC) conditions AGEPC receptors are lost from the plasma membrane and are reformed in the cells continuously.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Identification of receptors for platelet-activating factor in rat Kupffer cells.

Ligand binding studies demonstrated that isolated rat Kupffer cells possess high affinity binding sites for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC). AGEPC binding reached saturation within 10 min at 25 degrees C and was reversible. A Scatchard analysis revealed a single class of AGEPC receptors numbering about 10,600 sites/cell and possessing a dissociation constant of 0.45 nM. Similar values for the dissociation constant for AGEPC (0.12 and 0.34 nM) were obtained independently by kinetic analysis of specific AGEPC binding. AGEPC binding was stereospecific and was inhibited by Zn2+ and AGEPC receptor antagonists including BN52021 and U66985. The AGEPC receptor was functionally active since it was shown to mediate arachidonic acid release and eicosanoid production in Kupffer cells, and these events were inhibited by AGEPC receptor antagonist BN52021. The receptor-mediated arachidonic acid release was extracellular calcium-dependent and was abolished by calcium channel blocker prenylamine and by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, indicating that calcium influx through a receptor-regulated calcium channel in the plasma membrane is involved in the AGEPC-induced arachidonic acid release. It is suggested that rat Kupffer cells have specific and functionally active AGEPC receptors which are involved in signaling mechanisms which govern the production of several other autacoid-type mediators in the liver.

Animals↗

PubMed1989

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets.

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.

Arachidonic Acid↗

PubMed1989

Beta 2-adrenergic agonist regulation of immune aggregate- and platelet-activating factor-stimulated hepatic metabolism.

Vasoconstriction and subsequent glycogenolysis stimulated by immune complex infusion into perfused rat livers was inhibited by prior infusion of isoproterenol. Similarly, isoproterenol inhibited the biosynthesis of bioactive lipid autacoids such as platelet-activating factor, prostaglandin E2, and thromboxane B2 which was stimulated by immune aggregates. The adrenergic receptor specificity of these effects was determined through the use of specific adrenergic subtype-specific agonists and antagonists to be mediated by beta 2-adrenergic receptors. Indirect evidence for the differential expression of hepatic sinusoidal and parenchymal beta-adrenergic receptors in the male rat during ontogeny suggested that inhibition of immune aggregate-stimulated autacoid biosynthesis, vasoconstriction, and glycogenolysis by isoproterenol occurs at a sinusoidal locus, most likely Kupffer cells. In contrast with the ability of beta 2-adrenergic agonists to inhibit immune aggregate- and platelet-activating factor-stimulated hepatic metabolism, dibutyryl cyclic AMP did not mimic these sinusoidal beta 2-adrenergic effects, despite stimulating hepatic parenchymal cell glycogenolysis as effectively as isoproterenol. These observations suggest a role for cyclic AMP-independent mechanisms in the regulation of heterologous stimulus-response coupling by hepatic sinusoidal beta 2-adrenergic receptors.

Animals↗

PubMed1989

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.

The metabolism of platelet-activating factor (PAF; identified as AGEPC: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (lyso-GEPC: 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) was investigated in cultured rat Kupffer cells. The rat Kupffer cells accumulated [3H]AGEPC and deacetylated this compound to the corresponding [3H]lyso-GEPC, which was the major metabolic product of [3H]AGEPC. [3H]Lyso-GEPC was distributed primarily in the supernatant fraction of incubated cells throughout the experimental interval. Only a very small portion of the [3H]lyso-GEPC was further converted to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), indicating that this acylation process was not particularly active in these cells. When [3H]lyso-GEPC was incubated with Kupffer cells, the conversion of lyso-GEPC to AGEPC via the acetyltransferase reaction increased up to 30 min and declined thereafter. Bovine serum albumin (BSA) had a substantial influence on both the cellular uptake and the metabolism of [3H]AGEPC. An increase in the BSA concentration in the incubation media reduced the cellular uptake of [3H]AGEPC and the subsequent formation of lyso-GEPC. The results of this study suggest that the hepatic Kupffer cells play an important role in the metabolism of PAF. Moreover, these results infer that the regulation of the PAF level in certain hepatic pathophysiological situations may be a consequence of the production and subsequent metabolism of this potent lipid autacoid in the Kupffer cells of the liver.

Animals↗

PubMed1989

Biosynthesis of platelet-activating factor by cultured rat Kupffer cells stimulated with calcium ionophore A23187.

Cultured rat Kupffer cells synthesize and release platelet-activating factor (PAF) when stimulated with calcium ionophore A23187. The production of PAF is concentration- and time-dependent and, based upon [3H]serotonin release assays, approx. 1.0 pmol of PAF is formed per 8 x 10(6) cells during 10 min of ionophore stimulation. It is suggested that Kupffer cells are important cellular components which produce and release PAF in order to facilitate communication between hepatic sinusoidal and parenchymal cells. Further, it is suggested that such mediator production in response to reticulo-endothelial cell stimulation causes the hepatic glycogenolytic response previously in the isolated perfused rat liver.

Acetyl Coenzyme A↗

PubMed1989

Platelet-activating factor in human luteal phase endometrium.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is one of the most potent mediators of vascular permeability. PAF levels change in the rabbit endometrium just prior to implantation, which suggests that PAF may be a key substance transducing preimplantation embryonic signals. To study whether PAF was present in the human endometrium, and if so, to determine the cellular origin and hormonal regulation of endometrial PAF, specimens were obtained from 14 women (aged 23-42 yr) undergoing elective hysterectomy during the luteal phase of the cycle (plasma progesterone levels greater than 2 ng/ml). No specimens were taken from women with malignant uterine pathology. Stromal cells and epithelial glandular cells were separated by collagenase and DNAse digestion, and then cultured to confluence in vitro in medium 199. Radioimmunoassays of prostaglandin F (PGF) and prolactin in the culture media were used to confirm cell type and viability. PGF release into the culture medium from stromal cells was low (control 1.52 +/- 0.20 ng/ml), and unchanged by hormone treatment. In contrast, release of PGF from unstimulated glandular cells was 6.05 +/- 0.52 ng/ml, and was significantly increased (p less than 0.05) by estradiol or progesterone plus estradiol, to 12.17 +/- 1.67, and 8.60 +/- 0.81, respectively. Progesterone alone was without effect. Prolactin was secreted by stromal cell cultures, increasing steadily from 24 to 120 h. The levels in the medium were increased by progesterone. PAF activity was assessed by rabbit platelet aggregation and serotonin-release bioassays after lipid extraction and separation by thin-layer chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Production and effects of platelet-activating factor in the rat brain.

The synthesis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rat brain was evaluated. Extracted PAF was characterized using standard HPLC and TLC techniques, and by correlation of its bioactivity with the acetylation state of the 2-position of the molecule. PAF was quantified by bioassay, its ability to cause [3H]serotonin release from washed rabbit platelets. The low basal level of PAF (0.25 +/- 0.15 pmol/g wet wt., mean +/- S.E.) in the brain of the intact rat was greatly increased by intraperitoneal injection of the chemoconvulsant drugs picrotoxin or bicuculline, to levels of 10.68 +/- 2.18 and 4.97 +/- 0.75 pmol/g wet wt., respectively. Electroconvulsion also increased brain PAF, to 1.76 +/- 0.30 pmol/g wet wt. Equivalent experiments using bicuculline in the isolated perfused rat brain yielded qualitatively similar results, indicating that the production of PAF in the brain is independent of systemic metabolism. When a 32P-labeled nerve-ending (synaptosome) preparation from rat brain was challenged with synthetic PAF (denoted AGEPC) at 0.1 nM concentration, responses were observed consistent with accelerated turnover of polyphosphoinositides. AGEPC also caused an increase in the Na+-Ca2+ exchange of synaptic membrane vesicles. Furthermore, AGEPC infused into the vasculature of the isolated perfused rat brain caused changes consistent with an increase in blood-brain barrier permeability, although AGEPC did not itself significantly penetrate the blood-brain barrier. It is concluded from these studies that PAF is synthesized within the rat brain in response to convulsant stimuli and that one of its effects is to accelerate synaptic polyphosphoinositide turnover. In addition, circulating PAF can influence blood-brain barrier permeability without itself penetrating the blood-brain barrier.

Animals↗

PubMed1988

Location of platelet activating factor binding in rat liver.

Autoradiographs of tissue slices from livers perfused with 1 x 10(-9) M-1-O-[3H]octadecyl-2-acetyl-sn-glycero-3-phosphocholine ([ 3H]18:0-sn-3-AGEPC) indicate that binding of this agonist is localized in the portal venules in anterograde perfused livers, and in the central venules in retrograde perfused livers. The pattern of silver grains in anterograde perfused liver was not affected significantly by prior exposure to 100-fold excesses of unlabelled 16:0- or 18:0-sn-3-AGEPC, 16:0-sn-1-AGEPC, or a 1000-fold excess of U.66985. [3H]18:0-sn-3-lyso-GEPC produced the same pattern of binding as the acetylated analogue. Measurement of glucose release stimulated by 16:0-sn-3-AGEPC demonstrated that the retrograde perfused liver was nearly 1000-fold less sensitive to this compound than the anterograde perfused liver. Exposure of the livers to bovine serum albumin prior to 5 x 10(-11) M-[3H]18:0-sn-3-AGEPC resulted in inhibition of stimulated glucose release, and decreased both the amount of label retained in the livers and the amount of silver grains over the portal sinusoidal cells without affecting the amount of grains seen over all other regions of the liver. Glucose release from primary monolayer cultures of hepatocytes or suspensions of liver slices was not stimulated by 16:0-sn-3-AGEPC. The results suggest that specific binding of [3H]18:0-sn-3-AGEPC is restricted to the portal side of the liver microvasculature, the majority of binding is nonspecific, and the biological response to AGEPC requires an intact and perfused vasculature.

Animals↗

PubMed1988

Phospholipids chiral at phosphorus. Synthesis and stereospecificity of phosphorothioate analogues of platelet-activating factor.

RP and SP isomers of 1-O-hexadecyl-2-acetyl-3-thiophosphocholine (AGEPsC) have been synthesized. The activity of these isomers in platelet aggregation and serotonin secretion was compared with that of 1-O-hexadecyl-2-acetyl-3-phosphocholine (AGEPC). The results show that (SP)-AGEPsC has the same activity as AGEPC within experimental error in both assays. The RP isomer, however, is only 0.6-2% as active as AGEPC in platelet aggregation and serotonin release. The results suggest that the phosphate group of AGEPC is likely to be involved in the interactions with its receptor, at least in the events leading to platelet aggregation and secretion.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

AGEPC: a potent calcium-dependent chemical mediator in the liver.

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Animals↗

PubMed1988

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.

Animals↗

PubMed1988

Occurrence of platelet-activating factor in rabbit spermatozoa.

Spermatozoa obtained from rabbit ejaculate were analyzed for the presence of platelet-activating factor [PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)] by using standard HPLC and TLC procedures. Fractions corresponding to synthetic PAF (AGEPC) revealed PAF-like activity amounting to 0.35 +/- 0.06 pmol/10(8) cells (mean +/- SE) as determined by bioassays based on the release of [3H]serotonin from washed rabbit platelets. This activity was lost upon base-catalyzed methanolysis, but was restored to the original level after reacetylation. Analysis of the phosphatidylcholine (PC) fraction by GC-MS subsequent to base-catalyzed methanolysis showed that 1-O-alkyl-2-acylphosphocholine comprises about 12% of the PC fraction with alkyl chain lengths of 16:0 (88%) and 18:0 (12%).

Animals↗

PubMed1987

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.

Animals↗