Desensitization of human platelets by platelet activating factor

Human platelets are less responsive to PAF at 37 degrees than at 25 degrees. They can be desensitized to the effects of PAF by pre-exposure to small concentrations. In both cases desensitization appears to be accompanied by a decreased affinity of the high affinity site for PAF rather than loss of binding sites. Alteration of a metabolic step subsequent to binding cannot be excluded, but platelets show normal response to a variety of other agents under the conditions resulting in desensitization of platelets to PAF.     

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.     

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.     

Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

Bleomycin is an anti-tumor agent whose cytotoxicity is related to the introduction of both single-stranded and double-stranded breaks in cellular DNA. In an assay using isolated nuclei, low levels of ethidium bromide substantially increased bleomycin induced release of nuclear chromatin. Treatment of mouse L1210 leukemia cells in vitro with low levels of ethidium bromide followed 1 hr later by bleomycin produced a synergistic effect that was 8 fold greater than that expected from the additive cytotoxicity of each drug alone. Interestingly, when the order of drug addition was reversed the drug synergism was much reduced (2 fold). The combination of DNA unwinding and strand scission agents may represent a novel and rational approach to the chemotherapy of cancer.     

Ceramide does not inhibit protein kinase C beta-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) betaI and betaII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC beta-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change

The present study compares the molecular mechanism by which thrombin, platelet-activating factor, and epinephrine induce platelet activation. Thrombin and platelet-activating factor induce an initial activation of phospholipase C, as measured by formation of 1,2-diacylglycerol and phosphatidic acid, during platelet shape change which is independent of and dissociated from metabolism of arachidonic acid. Phospholipase C activation and shape change are independent of extracellular Ca2+ and Mg2+. Formation of cyclooxygenase products occurs subsequent to the initial activation of phospholipase C and those metabolites are associated with platelet aggregation and further activation of phospholipase C. On the other hand, epinephrine is an unique platelet stimulus since it requires extracellular divalent cations and does not induce platelet shape change or activation of phospholipase C. Our results indicate that activation of phospholipase C may be a mechanism by which physiological agonists can activate platelets independently of extracellular divalent cations.     

Phosphatidylcholine synthesis in platelets is stimulated by diacylglycerol but not by phorbol ester

The biosynthesis of phosphatidylcholine (PC) in platelets was followed by measuring the incorporation of 32Pi. Incorporation into PC was stimulated by treatment with Clostridium perfringens phospholipase C or with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. However, neither the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate or thrombin stimulated 32Pi incorporation into PC. We conclude that phorbol ester does not stimulate the hydrolysis of PC to diacylglycerol in platelets.     

Biosynthesis of platelet activating factor in rabbit polymorphonuclear neutrophils

The synthesis of platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied in rabbit peritoneal polymorphonuclear neutrophils. Upon stimulation with ionophore A23187 and Ca2+, these cells are able to incorporate [3H]acetate or 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine into platelet activating factor. Under the same incubation conditions, however, the cells do not synthesize platelet activating factor from [14C]hexadecanol, which is an immediate precursor of O-alkyl chains in the de novo pathway. In the absence of ionophore, [14C] hexadecanol is incorporated into 1-O-alkyl-2-acyl-sn-glycerol-3-phosphate and subsequently into the 1-O-alkyl-linked choline and ethanolamine phosphoglyceride pools. However, in the presence of ionophore, [14C] hexadecanol incorporation is limited to phosphatidic acid, perhaps due to the inhibition of choline phosphotransferase. These findings provide strong evidence that platelet activating factor is synthesized by a deacylation-reacylation mechanism. Upon stimulation, these cells can utilize both plausible substrates of this pathway to make the final product, while under the same conditions it appears that a key step of the de novo pathway is inhibited.     

Activation of rabbit platelet phospholipase and thromboxane synthesis by 1-o-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor)

1-O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), structurally identical with platelet activating factor, is a potent stimulus for rabbit platelet aggregation and serotonin secretion. AGEPC at concentrations between 10⁻¹⁰ and 10⁻⁸ M induced stimulation of rabbit platelet synthesis of thromboxane B₂. The dose vs. response curve for platelet thromboxane B₂ synthesis was displaced slightly towards higher stimulus concentrations compared to [³H]serotonin secretion, with half-maximal concentrations of 2.5 · 10⁻⁹ and 8 · 10^t-10 M, respectively. Rates of thromboxane B₂ synthesis and secretion were similar with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>max</mtext>}}$ of 4.0–4.5 s for both processes. AGEPC induced a decrease in platelet [¹⁴C]arachidonic acid in both phosphatidylinositol and phosphatidylcholine, although [¹⁴C]arachidonic acid turnover in phosphatidylcholine was not observed below 1 · 10⁻⁸ M AGEPC. Concomitantly, this decrease in phospholipid [¹⁴C]arachidonic acid was associated with a marked increase of radiolabel in platelet diacylglycerol and phosphatidic acid 15s after AGEPC addition, suggesting the possibility of a phospholipase C-diacylglycerol lipase mechanism of fatty acid cleavage. As observed previously with secretion and aggregation, removal of the 2-acetyl group from AGEPC abrogated all capacity of this molecule to stimulate platelet phospholipase. This study indicates that AGEPC (or platelet activating factor) activation of rabbit platelet phospholipase occurs in a time-course and concentration range similar to that required for [³H] serotonin secretion.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

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.     

Ceramide does not inhibit protein kinase C β-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) βI and βII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC β-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Dexamethasone inhibits plasminogen activator activity in experimental pemphigus in vivo but does not block acantholysis

In vitro studies have suggested that autoantibody-stimulated increases in epidermal plasminogen activator (PA) may be an important pathogenetic mechanism in pemphigus vulgaris (PV). We measured PA in murine epidermis after i.p. injection of normal human IgG (NH IgG) and PV IgG, with and without exposure to dexamethasone (DEX). BALB/c neonates received i.p. injections of saline control or DEX (20 mg/kg). Twenty-four hours later, they received a second injection of saline or DEX and a single dose of NH or PV IgG (20 mg/gm body weight). After 24 hr, epidermis was obtained and was sequentially extracted in 0.14 M NaCl, pH 6.8, and 0.5% Triton X-100 in 0.1 M Tris, pH 8.1. Epidermal PA was assayed in the Triton-Tris supernatant by a two-stage colorimetric reaction and was expressed as milliPloug units per milligram of protein (mPu/A280). PA in animals injected with NH IgG was 0.21 +/- 0.11 mPu/A280 (n = 8). Epidermal PA was increased in animals with cutaneous lesions of pemphigus to 0.42 +/- 0.29 (n = 15). Treatment with DEX decreased PA levels in both animals receiving NH IgG and PV IgG by 80%, to 0.04 +/- 0.05 (n = 15) and 0.09 +/- 0.07 (n = 7), respectively. Despite the decreased PA activity, all animals in the PV IgG and the PV IgG-plus-DEX group had identical and extensive cutaneous disease, and lesions developed at the same time points. This finding shows that PV autoantibodies can stimulate increases in epidermal PA, but reduction of PA by corticosteroids does not inhibit acantholysis in vivo. There is no clear correlation between PA and disease activity in the murine model of pemphigus.     

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.     

Protein kinase C potentiates corticotropin releasing factor stimulated cyclic AMP in pituitary

The hypophysiotrophic hormone corticotropin releasing factor (CRF) stimulates the anterior pituitary corticotroph to export stress hormones such as adrenocorticotrophic hormone (ACTH). In rat anterior pituitary cells, CRF-induced elevation of cyclic AMP was profoundly potentiated (by an order of magnitude) by stimulators of protein kinase C. This effect occurred within minutes, was concentration dependent, and exhibited the appropriate pharmacological specificity to attribute the effects to protein kinase C. Phorbol myristate acetate (PMA), phorbol dibutyrate (PDB) and teleocidin were active with appropriate EC50's, while 4-alpha-PMA was inactive. PMA and PDB were also ACTH secretagogues in their own right. We suggest that protein kinase C can modulate CRF receptor coupling to the adenylate cyclase holoenzyme in anterior pituitary cells.     

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [³²P]orthophosphate or myo-[³H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of ³²P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.