Purification and characterization of human platelet phospholipase A2 which preferentially hydrolyzes an arachidonoyl residue

A phospholipase A2 with an arachidonoyl residue preference was purified about 11,700-fold from human platelet soluble fraction to near homogeneity. The purified phospholipase A2 exhibited a molecular mass of about 90 kDa on SDS polyacrylamide gel electrophoresis and hydrolyzed phospholipids with an 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 x 10(-7) and 10(-6) M free calcium ion. Thus, the 90-kDa phospholipase A2 is considered to be a novel enzyme, distinct from the 14-kDa one previously purified from human platelets. The 90-kDa phospholipase A2 may participate mainly in arachidonate metabolism of platelets.     

Immunochemical detection of arachidonoyl-preferential phospholipase A2.

Monoclonal antibodies were raised against rabbit platelet cytosolic arachidonoyl-preferential phospholipase A2. The antibodies precipitated the arachidonoyl-preferential phospholipase A2 activity in the soluble fraction of a rabbit platelet lysate in combination with an immobilized anti-mouse immunoglobulin antibody, and reacted predominantly with a protein exhibiting a molecular weight of approximately 88,000 on immunoblotting analysis. All three antibodies established so far reacted with human platelet arachidonoyl-preferential phospholipase A2 as effectively as the rabbit platelet enzyme. One of them reacted with the rat platelet arachidonoyl-preferential enzyme, whereas none of them reacted with rabbit platelet secretory 14-kDa group II phospholipase A2. The existence of an immunologically related phospholipase A2 was further shown in rabbit granulocytes, brain, lung, and liver, rat and mouse mast cells, and human monocytoma U937 cells. Thus, an arachidonoyl-preferential phospholipase A2 with similar structural properties appeared to be expressed in a variety of cells and tissues.     

Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.     

Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization

We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location.     

Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.     

Purification and characterization of a soluble phospholipase A2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.     

Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis

Extracellular phospholipase A2 was purified about 1.7 X 10(5) fold to near homogeneity from human synovial fluid of rheumatoid arthritis by sequential use of column chromatographies on heparin-Sepharose, butyl-Toyopearl, and reversed-phase HPLC. The final preparation showed a single band on SDS-polyacrylamide gel electrophoresis, and its molecular mass was estimated to be approximately 13,700 daltons. The purified enzyme had a pH optimum of 9.0 and required Ca2+ for maximum activity. It hydrolyzed phosphatidyl-ethanolamine more effectively than phosphatidylserine and phosphatidylcholine. These properties were similar to those of an extracellular phospholipase A2 detected in the peritoneal cavity of caseinate-treated rats.     

广西眼镜王蛇毒碱性磷脂酶A_2(PLA_2)的分离纯化及其性质的研究

广西眼镜王蛇毒用羧甲基纤维素CM-52、磷酸纤维素P-11和Sepharose CL-6B柱层析纯化,得到一个在聚丙烯酰胺凝胶电泳上为单一蛋白带,PLA_2的比活性较原蛇毒提高3.6倍,分子量的为13000,由122个氨基酸组成,_pI为8.9,具有良好的热稳定性。从碱性PLA_2对红细胞影响的电镜观察可见,对人的红细胞膜有明显的作用,而对山羊红细胞作用不明显。PLA_2无论对人还是对山羊、兔和豚鼠红细胞电泳速度都有明显的迟缓作用。     

Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas

A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.     

The phospholipase A2 of human spermatozoa; purification and partial sequence.

In view of its proposed key role in the acrosome reaction, phospholipase A2 has been isolated and purified from human spermatozoa. Following SDS-PAGE, a single major band was obtained with an estimated molecular mass of 16.7 kDa. Sequence analysis of the N-terminal portion of the molecule revealed the identity of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. From this partial analysis it is evident that the phospholipase A2 of human spermatozoa represents a new sequence. Of interest is the location of glutamine-4, phenylalanine-5, methionine-8 and isoleucine-9; this sequence appears to be highly conserved throughout evolution.     

Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus

Antibodies against Bacillus cereus phospholipase C were prepared in rabbits and used to affinity purify a phosphatidylcholine-preferring phospholipase C from a human monocytic cell line. Affinity chromatography resulted in an approximately 3000-fold, one-step enrichment of phospholipase C. The human enzyme had an apparent molecular mass of 40,000 daltons as determined by SDS gel electrophoresis. Western blotting analysis demonstrated that this protein interacted specifically with the rabbit antibody raised against bacterial phospholipase C. The purified enzyme preferred phosphatidylcholine as a substrate, was neutral pH active and was inhibited by EGTA. These studies demonstrate that antibodies raised against bacterial phospholipase C may be useful in purifying phospholipase C from a human source.     

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.     

Identification of a calcium-independent phospholipase A2 in rat lung cytosol and differentiation from acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether)

The 100 000 X g supernatant of total rat lung homogenate was found to contain at least three phospholipase A2-type activities. Gel filtration separated a low molecular weight and Ca2+-requiring phospholipase A2 from Ca2+-independent acylhydrolase peak with an apparent higher molecular weight. Upon DEAE-cellulose chromatography this fraction was separated into a Ca2+-independent acylhydrolase and a Ca2+-independent platelet-activating factor-acetylhydrolase with no apparent overlap in acyl chain length specificity. The long-chain acylhydrolase was shown to exhibit specificity for the ester bond at the sn-2-position. Ca2+-independent phospholipase A2 activity was inhibited by p-bromophenacylbromide and was resistant to diisopropylfluorophosphate. In contrast, the Ca2+-independent acetylhydrolase activity was inhibited by diisopropylfluorophosphate but was unaffected by p-bromophenacylbromide.     

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.     

Hydrolysis of 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine by human pancreatic phospholipase A2

A novel fluorescent phospholipid analogue, 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (C30PHPC) was employed as a substrate for human pancreatic phospholipase A2. C30PHPC has a main endothermic phase transition with Tm at 46 degrees C as determined by differential scanning calorimetry (DSC). For an aqueous dispersion of C30PHPC the ratio of the intensities of pyrene excimer and monomer fluorescence emission, (IE/IM) has a maximum between 32 and 36 degrees C. The excimer emission intensity (at 480 nm) exceeds the monomer emission intensity (at 400 nm) 6.5-fold thus indicating a close packing of the phospholipid pyrene moieties in the lipid phase. C30PHPC has a limiting mean molecular area of 37 A2 at surface pressure 35 dyn cm-1 as judged by the compression isotherm at an air-water interphase. The hydrolysis of C30PHPC by human pancreatic phospholipase A2 was followed by monitoring the increase in the pyrene monomer fluorescence emission intensity occurring as a consequence of transfer of the reaction product, pyren-1-yl hexanoic acid into the aqueous phase. The enzyme reaction exhibited an apparent Km of 2.0 microM substrate. Calcium at a concentration of 0.2 mM activated the enzyme 4-fold. Maximal hydrolytic rates were obtained at 45 degrees C and at pH between 5.5 and 6.5. The enzyme reaction could be inhibited by 5 mM EDTA, confirming the absolute requirement for Ca2+ of this enzyme. The present fluorimetric assay easily detects hydrolysis of C30PHPC in the pmol min-1 range. Accordingly, less than nanogram levels of human pancreatic phospholipase A2 can be detected.     

Studies on the acyl-chain selectivity of cellular phospholipases A2

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 A2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[14C]oleoylphosphatidylcholine and sn-2-[3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A2 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 A2 are discussed.     

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.     

Biochemical characterization of phospholipase D activity from human neutrophils

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol-1-phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2(+)-independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350-400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was detected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal-transducing processes during cell stimulation in human phagocytes.     

Purification and properties of phospholipase A2 from human seminal plasma

The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.     

Secretory phospholipase A2 enzymes in atherogenesis

PURPOSE OF REVIEW: Immunohistochemistry studies have confirmed the presence of group IIA, group V and group X secretory phospholipase A2 in human or mouse atherosclerotic lesions. The possibility that secretory phospholipase A2 plays a role in the pathophysiology of atherosclerosis (and is not merely a marker for localized inflammation) has been substantiated by a number of recent in-vitro and in-vivo studies. RECENT FINDINGS: A mouse strain with a targeted deletion of group V secretory phospholipase A2 has been developed. Peritoneal macrophages from these mice have significantly blunted eicosanoid generation in response to zymosan, providing the first direct evidence that a secretory phospholipase A2 plays a role in stimulation-induced arachidonic acid production in vivo. A recent in-vitro study indicated that de novo synthesized groups IIA and X secretory phospholipase A2 can mediate arachidonic acid release intracellularly, without the requirement for previous secretion from cells, as was previously thought. Several studies support the previously proposed model that secretory phospholipase A2 hydrolysis generates pro-atherogenic LDL. These data, coupled with the finding that macrophage-specific expression of human group IIA secretory phospholipase A2 promotes atherosclerotic lipid deposition in mice, draw attention to secretory phospholipase A2 as an attractive target for the treatment of atherosclerotic disease. SUMMARY: Secretory phospholipase A2 activity in the arterial intima has the potential to amplify atherogenic processes by liberating potent pro-inflammatory lipid mediators and by generating pro-atherogenic LDL. Future in-vivo studies will aid in defining the mechanism(s) that underlie the pro-atherosclerotic effects of secretory phospholipase A2.