Human Platelet Phospholipase Research Articles

Lineage-specific defect in gene expression in human platelet phospholipase C-β2 deficiency

AbstractPhospholipase C (PLC)–β2 plays a major role in platelet activation. Previous studies have described a unique patient with impaired receptor-mediated platelet aggregation, secretion, calcium mobilization, and phospholipase C (PLC) activation associated with a selective decrease in platelet PLC-β2 isozyme. To identify the mechanisms leading to the defect, platelet RNA from the patient and healthy subjects was subjected to reverse transcription–polymerase chain reaction (RT-PCR) and the products sequenced. The PLC-β2 cDNA sequence in the patient showed no abnormalities. Platelet PLC-β2 and β-actin (internal control) mRNA levels were assessed by RT-PCR; the ratio of PLC-β2 to β-actin mRNA levels was 0.80 to 0.95 in 4 healthy subjects and 0.28 in the patient. PLC-β2 mRNA levels were similarly reduced compared with GPIIb and Gαq mRNA levels. PLC-γ2 and platelet factor 4 mRNA levels were normal. Calcium mobilization was studied in neutrophils upon activation with formyl-Met-Leu-Phe (fMLP), adenosine diphosphate (ADP), platelet-activating factor (PAF), interleukin-8 (IL-8), C5a, and leukotriene B4 (LTB4), and it was normal. Neutrophil elastase secretion upon activation with fMLP, ADP, PAF, IL-8, C5a, and LTB4 was normal, as were neutrophil PLC-β2 mRNA and PLC-β2 on immunoblotting. Thus, responses to activation, PLC-β2 protein, and PLC-β2 mRNA are decreased in patient platelets but not in neutrophils, providing evidence for a hitherto undescribed lineage (platelet)–specific defect in PLC-β2 gene expression. These studies provide a physiologically relevant model to delineate regulation of PLC-β2 gene and its tissue-specific expression.

Biaryl diacid inhibitors of human s-PLA2 with anti-inflammatory activity

Twenty-four hydrophobic dicarboxylic acids are described which were evaluated as inhibitors of 14kDa human platelet phospholipase A2 (HP-PLA2). In general, biarylacetic acid derivatives were found to be more active than biaryl acids or biarylpropanoic acids. More potent inhibitors were obtained when hydrophobic groups were attached to the biaryl acid nucleus using an olefin linkage as compared to an ether linkage. Compounds with larger hydrophobic groups were usually more potent inhibitors of HP-PLA2. Five of the compounds disclosed in this report (2, 4, 28, 36b and 36i) were found to possess significant anti-inflammatory activity in a phorbol ester induced mouse ear edema model of chronic inflammation.

A simple synthesis of biaryl phospholipase A 2 inhibitors: Probing hydrophobic effects

A series of biaryl acids has been synthesized by treating diphenic anhydride with a variety of amines. The one step strategy allows the rapid interchange of groups for evaluating the hydrophobic constraints of inhibitors of phopholipase A 2. The most active inhibitor synthesized displayed an IC 50 = 6 μM against human platelet phospholipase A 2.

Synthesis of lipidic amino acid and dipeptide inhibitors of human platelet phospholipase A2

Phospholipases A2 (PLA2s) catalyze the hydrolysis of glycerophospholipids at the sn-2 position, and their inhibition is considered a rational approach for the prevention and treatment of inflammation. A number of amides and esters of alpha-amino acids with saturated linear side chains, esters of alpha-amino alcohols and derivatives of lipidic peptides were prepared and tested against secreted humal platelet phospholipase A2. Among the compounds tested the amides of free amino acids with long-chain amines presented the highest in vitro inhibitory activity.

Lipid mimetics as inhibitors of human platelet phospholipase A2.

Lipid mimetics as inhibitors of human platelet phospholipase A2.

Synthesis of medicinally useful lipidic?-amino acids, 2-amino alcohols and diamines

The lipidicα-amino acids (LAAs) are non-naturalα-amino acids with saturated or unsaturated long aliphatic side chains. LAAs and their derivatives (lipid mimetics) together with the lipidic peptides represent a class of compounds which combine structural features of lipids with those of amino acids and peptides. Racemic LAAs may be prepared by classical methods and resolved by chemical or enzymatic methods. LAA amides and esters with saturated or unsaturated long chain amines and alcohols respectively, as well as lipidic dipeptide derivatives inhibit both pancreatic and human platelet phospholipase A2. Lipophilic peptide derivatives are inhibitors of human neutrophil elastase. LAAs and their oligomers have been used as drug delivery system. A Lipid-Core-Peptide system has been designed and used as a combined adjuvant-carrier-vaccine system. A variety of lipid mimetics such as lipidic 2-amino alcohols, lipidic 1,2- and 1,3-diamines have been prepared based upon LAAs. Some of them are potent inhibitors of phospholipase A2. A general approach to enantioselective synthesis of LAAs and lipid mimetics is based on the oxidative cleavage of 3-amino-1,2-diols obtained by the regioselective opening of enantiomerically enriched long chain 2,3-epoxy alcohols.

The Platelet-Stimulating Effect of Adrenaline Through α2-Adrenergic Receptors Requires Simultaneous Activation by a True Stimulatory Platelet Agonist

The stimulating effect of adrenaline on human platelet phospholipase C (PLC) activation and responses in vitro (shape change, aggregation and dense granule secretion) was investigated with respect to its dependence on exogenously added agonists. All experiments were performed with human gel-filtered platelets pretreated with acetylsalicylic acid to prevent endogenous stimulation by the arachidonate pathway. (1) Preliminary experiments demonstrated the presence of trace amounts of extracellular ADP (0.05-0.58 microM) in non-stimulated platelet suspensions; ADP was effectively converted to ATP by the enzyme system creatine phosphate (CP)/creatine phosphokinase (CPK). (2) The adrenaline-induced optical aggregation and single particle (platelet) disappearance in the presence of trace amounts of ADP were almost abolished by the ADP-scavenger system CP/CPK. (3) The response of CP/CPK-treated thrombin- or platelet-activating factor (PAF)-stimulated platelets was markedly increased by a subsequent addition of adrenaline. When hirudin or BN 50726 was added just prior to adrenaline to terminate the activation by thrombin or PAF, respectively, the stimulating effect of adrenaline was also abolished. (4) CP/CPK-treated, PAF-stimulated platelets rapidly developed decreased responsiveness to a subsequent addition of PAF. When adrenaline was added instead of a second addition of PAF, the stimulating effect of adrenaline was gradually decreased and prevented in parallel with the homologous desensitization of PAF. (5) The weak platelet agonist serotonin by itself induced only shape change in CP/CPK-treated platelets. Adrenaline failed to enhance the extent of this serotonin-induced platelet activation. (6) These results strongly suggest that adrenaline per se is not a platelet agonist in vitro but acts to enhance the stimulation induced by true agonists.

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A 2 by acylamino phospholipid analogues

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A 2 (PLA 2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA 2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249–257]. Both the mutant platelet PLA 2 and the human pancreatic PLA 2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA 2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA 2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA 2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14–16 carbon atoms on position two. Because the platelet PLA 2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.

Purification and characterization of human platelet phospholipase A 2 which preferentially hydrolyzes an arachidonoyl residue

A phospholipase A 2 with an arachidonoyl residue preference was purified about 11 700-fold from human platelet soluble fraction to near homogeneity. The purified phospholipase A 2 exhibited a molecular mass of about 90 kDa on SDS polycrylamide gel electrophoresis and hydrolyzed phospholipids with a arachidonoyl residue more effectively than those with a linoleoyl residue. The catalytic activity of the purified enzyme detected with phosphatidylcholine as a substrate increased sharply between 3 × 10 −7 and 10 −6 M free calcium ion. Thus, the 90-kDa phospholipase A 2 is considered to be a novel enzyme, distinct from the 14-kDa one previously purified from human platelets. The 90-kDa phospholipase A 2 may participate mainly in arachidonate metabolism of platelets.

Activation of Human Platelet Phospholipase C by Calpain-I

Effects of calpain-I purified from human erythrocytes on phosphatidylinositol hydrolysis activity of phospholipase C (PI-PLC) were investigated by using human platelet lysate and the partial purified enzymes. PI-PLC activity in human platelet lysate prepared in the absence of thiolprotease inhibitors (leupeptin and EST) was higher than that prepared in the presence of inhibitors. This result implied that PI-PLC was activated by thiolprotease in platelets during preparation of platelet lysate. When the thiolprotease inhibitors were removed by gel filtration from platelet lysate prepared in the presence of the inhibitors and calpain-I was added into the preparation, the PI-PLC activity was enhanced in correlation with the amount of calpain-I. PI-PLC in platelets lysate was partially purified by phenyl-Sepharose and Ultrogel AcA22 column chromatography. PI-PLC was separated to two isozymes by phenyl-Sepharose chromatography. The apparent molecular weight of these isozymes was about 200kDa in AcA22 gel filtrations. One of the PI-PLC, which was adsorbed with phenyl-Sepharose column and eluted with the buffer containing 50% ethyleneglycol, was not influenced by the preincubation with calpain-I, but the activity of the other one, which was not adsorbed with phenyl-Sepharose column, was enhanced three times by the preincubation with calpain-I. These results indicate that two isozymes of PI-PLC are located in platelets and one of the enzymes is activated by calpain-I.

Stimulation of phospholipase A2 activity in human platelets by trypsin and collagen

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of type I collagen on platelet membrane phospholipase A2 activity was also observed in a cell-free system utilizing platelet membranes. The release of arachidonic acid was enhanced by types I and III but not by type V collagen. The activation of platelet phospholipase A2 by type I collagen was inhibited by soybean trypsin inhibitor and mimicked by trypsin. However, type I collagen addition was not associated with any detectable changes in platelet membrane proteins while trypsin altered many proteins. These results point to acid soluble phospholipase A2 activity of platelets as an enzyme activated by type I collagen.

Studies on the acyl-chain selectivity of cellular phospholipases A 2

The selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A 2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A 2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[ 14 C] oleoylphosphatidylcholine and sn-2-[ 3 H] arachidonoylphosphatidylcholine either as single substrates or in doub labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A 2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A 2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A 2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-2-linoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A 2 are discussed.

Detection in human platelets of phospholipase A2 activity which preferentially hydrolyzes an arachidonoyl residue.

It has been generally considered that highly specific liberation of arachidonic acid is induced upon the stimulation of the platelets, although the molecular mechanism of the regulation of its action has not been well understood. An aim of the present study is to clarify the role of phospholipase A2 in the arachidonic acid metabolism within human platelets. Phosphatidylcholine or phosphatidylethanolamine with arachidonate at the sn-2 position of glycerol was cleaved efficiently by phospholipase A2 activity in homogenates as well as in the cytoplasmic fraction of human platelets, leading to the selective liberation of free arachidonate, whereas phospholipids with linoleate were hardly hydrolyzed under the same conditions. Double-reciprocal plots of kinetic data further strengthened the conclusion that human platelet phospholipase A2 showed high selectivity for arachidonoyl residue. This enzyme may play a crucial role in the intracellular metabolism of the arachidonate of phospholipids.

Human platelet phospholipase A 2 activity is responsive in vitro to pH and Ca 2+ variations which parallel those occurring after platelet activation in vivo

Human platelet phospholipase A 2 activity is responsive in vitro to pH and Ca 2+ variations which parallel those occurring after platelet activation in vivo

Human platelet phospholipase A 2 activity is responsive in vitro to pH and Ca 2+ variations which parallel those occurring after platelet activation in vivo

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A 2 enzyme species whose activity is regulated by Na +/H + exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660–8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197–9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na +/H + antiporter enables the phospholipase A 2 enzyme to function at ambient or low concentrations of intraplatelet Ca 2+. To test the hypothesis that the Ca 2+ dependence of platelet phospholipase A 2 activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca 2+ dependence of this enzyme as a function of changes in pH (from pH 6.8–8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A 2 enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca 2+ (EC 50 1–2 μM) and plateaued above 10 μM Ca 2+. Enzymatic activity measured at 4.8 μM Ca 2+ was increased by raising the pH from 5.5 to 8.0 (EC 50 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca 2+ concentrations. The interdependent regulation of phospholipase A 2 activity by changes in pH and Ca 2+ suggests that phospholipase A 2 could serve to integrate changes in intracellular pH and available Ca 2+ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.

Inhibition of human platelet phospholipase A2 by mono(2-ethylhexyl)phthalate.

There is evidence that the carcinogenic and teratogenic effects attributed to the plasticizer di(2-ethylhexyl)phthalate (DEHP) are due to its major metabolite mono(2-ethylhexyl)phthalate (MEHP). MEHP is also formed ex vivo by a plasma enzyme in blood products stored in polyvinyl chloride (PVC) DEHP plastic containers. People who receive large amounts of blood products, such as hemophiliacs or patients undergoing hemodialysis, cardiopulmonary bypass, or massive transfusion, are exposed to significant levels of plasticizer. In this study, the platelet was used to show that MEHP inhibits phospholipase A2 (PLA2), one of enzymes important in the release of arachidonic acid from membrane phospholipids. Arachidonate is the parent molecule for the synthesis of prostaglandins, thromboxanes, leukotrienes, and lipoxins that are made by a wide variety of cells. PLA2 was measured by the liberation of 14C-arachidonic acid from 1-stearoyl-2-[1-14C]arachidonyl-L-3-phosphatidylcholine. MEHP inhibits PLA2 activity noncompetitively in intact human platelets and lysates with a Ki of 3.7 x 10(-4) M. DEHP does not inhibit PLA2 in whole platelets. Inhibition of PLA2 by MEHP occurs at only three times the circulating level of MEHP measured in neonates undergoing exchange transfusion and 20-fold the levels experienced by patients during cardiopulmonary bypass. Therefore, infants and adult patients with multisystem failure who accumulate MEHP in their blood may be at risk for decreased platelet function.

Inhibition of Human Platelet Phospholipase A 2 by Mono(2-Ethylhexyl)phthalate

Inhibition of Human Platelet Phospholipase A 2 by Mono(2-Ethylhexyl)phthalate

Hydrolysis of 1-alkyl-2-arachidonoyl- sn-glycero-3-phosphocholine, a common precursor of platelet-activating factor and eicosanoids, by human platelet phospholipase A 2

The metabolism of platelet-activating factor (PAF) and arachidonic acid is linked through the common intermediate 1-alkyl-2-arachidonoyl- sn-glycero-3-phosphocholine (alkylarachidonoyl-GPC). Hydrolysis of alkylarachidonoyl-GPC by phospholipase A 2 may initiate the biosynthesis of both PAF and eicosanoids, since alkyllyso-GPC is formed for acetylation to PAF and arachidonic acid is liberated for conversion to biologically active metabolites. In order to elucidate the regulation and functional role of human platelet phospholipase A, in the pathway leading to the formation of both classes of lipid mediators, we have characterized its action upon alkylarachidonoyl-GPC. Human platelet phospholipase A, was solubilized and then partially purified in the presence of n- octyl-β- d- glucopyranoside (octyl glucoside). Hexadecylarachi-donoyl-GPC was prepared biosynthetically using platelet sonicates, purified by two-step high-performance liquid chromatography (HPLC) and suspended in buffer by sonication. Our results indicate that deacylation of alkylarachidonoyl-GPC by platelet phospholipase A 2 has an absolute requirement for Ca 2+. It occurs at submicromolar concentrations of free Ca 2+ and exhibits a biphasic Ca 2+-dependence with activity plateaus at 10 μM and 2 mM. Phospholipase A 2-mediated hydrolysis of alkylarachidonoyl-GPC is increased 2-fold by albumin and is enhanced 5-fold if 1,2-dioleoylglycerol is incorporated into the substrate dispersion. The substrate dependence and specificity of platelet phospholipase A 2 for 1-alkyl- vs. 1-acyl-linked subclasses of arachidonic acid containing phosphatidylcholine was examined with 1- O-hexadecyl-2-arachidonoyl- sn-glycero-3-phosphocholine (hexadecylarachidonoyl-GPC) and l-palmitoyl-2-arachidonyl- sn-glycero-3-phosphocholine (palmitoylarachidonoyl-GPC). We found that the substrates were deacylated equivalently. We conclude that, in stimulated platelets, in the presence of increased levels of cytoplasmic free Ca 2+ and newly generated diacylglycerol, alkylarachidonoyl-GPC may be rapidly hydrolyzed by phospholipase A 2 and may serve as a precursor of both PAF and eicosanoids.

Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol.

We examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of n-octyl beta-D-glucopyranoside (octyl glucoside), and its enzymic activity was determined with sonicated 2.5 microM-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (arachidonoyl-PC) as substrate. Phospholipase A2 activity was increased when diacylglycerol was incorporated into the substrate arachidonoyl-PC. Stimulation was maximal in the presence of greater than or equal to 29 mol% (1 microM) diacylglycerol, and was greater than 4-fold for both 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonoylglycerol. 1-Stearoyl-2-arachidonoylglycerol at concentrations of 2-5 mol% increased phospholipase A2 activity 1.3-1.8-fold. Exogenously added 1-oleoyl-2-acetylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 microM. Comparative studies conducted with pancreatic, bee-venom and snake-venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but stimulation was less than observed for platelet phospholipase A2. Our results suggest that diacylglycerol, known to be generated in stimulated platelets, may enhance Ca2+-activated phospholipase A2.