Calmodulin stimulates human platelet phospholipase A2.

Calmodulin is a ubiquitous Ca²⁺-binding protein, mediating the effect of Ca²⁺ on many enzyme systems and cellular reactions. Phospholipase A₂ (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) which governs the level of arachidonic acid in human platelets, requires Ca²⁺ for maximum activity. Results presented herein suggest that the stimulation of phospholipase A₂ by Ca²⁺ is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin.     

Evidence for two forms of phospholipase A2 in human semen

The molecular weight of the active unit of phospholipase A₂ (PA₂) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA₂ activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA₂ activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA₂ was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA₂ activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA₂ when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA₂ was found and was sensitive to changes in temperature.     

Solubilization and properties of Ca2+-dependent human platelet phospholipase A2

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.     

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

The adenosine analogues 5'-(N-ethyl)carboxamidoadenosine (NECA) and N6-(phenylisopropyl)adenosine (PIA) activate glycogen phosphorylase 5-fold and 4.2-fold respectively in rat hepatocytes incubated in the absence of endogenous adenosine. Half-maximally effective concentrations are 0.5 microM for NECA and 20 microM for PIA, demonstrating the presence of A2-adenosine receptors. Exogenous adenosine activates phosphorylase 4.6-fold, but high rates of adenosine disappearance from the medium render estimates of its half-maximally effective concentration unreliable. These effects of NECA and adenosine are inhibited by 0.1 mM-caffeine. Activation of phosphorylase by a physiological concentration of adenosine (3.3 microM) was 50% inhibited by a physiological concentration of caffeine (35 microM).     

Vitamin E inhibits platelet phospholipase A2

One of the most important functions of phospholipase A2 is the release of arachidonic acid from membrane phospholipids for the synthesis of biologically active eicosanoids. We have demonstrated in our laboratory that vitamin E inhibits platelet phospholipase A2 in a dose-dependent manner. Rats fed a 100 ppm or a 1000 ppm vitamin E diet exhibit diminished phospholipase A2 activity compared to those fed a vitamin E-free diet. Addition of vitamin E to a sonicated platelet suspension resulted in further suppression of the phospholipase A2 activity in all groups of rats. In order to gain insight into the mechanism of vitamin E inhibition of platelet phospholipase A2, we partially purified this enzyme by gel filtration chromatography. Enzyme activity was localized in the soluble supernatant fraction of a high-speed spin. This partially purified rat platelet phospholipase A2 had an absolute requirement for Ca2+ and was inhibited by various forms of tocopherol. Tocol inhibited the enzyme to a greater extent than either D- or DL-alpha-tocopherol, while there was little or no effect from DL-alpha-tocopherol acetate. These results emphasize the importance of the hydroxyl moiety on the chromanol of the vitamin E molecule for its inhibitory action, compared to that of the methyl groups which are absent in tocol. This inhibitory action of vitamin E on platelet phospholipase A2 suggests a crucial function for vitamin E in regulating arachidonate release from the membrane phospholipids and its subsequent metabolism.     

Monoclonal antibodies against rat platelet phospholipase A2

Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2.     

The primary structure of rat platelet phospholipase A2

In our previous report (Hayakawa, M., Kudo, I., Tomita, M., & Inoue, K. (1988) J. Biochem. 103, 263-266), we have shown that phospholipases A2 purified from rat platelet membrane fractions and an extracellular medium of thrombin-stimulated rat platelets were essentially identical to each other. Both purified enzymes were digested with proteases, and the resulting peptides were subjected to primary sequence determination. The sequence analysis of the HPLC-separated peptides and the alignment of the sequences showed a tentative primary structure of rat platelet phospholipase A2, which was composed of 125 amino acid residues. It showed 47% homology with snake venom Agkistrodon halys blomhoffii phospholipase A2.     

Multiple forms of secretory phospholipase A2 in plants

Multiple secretory phospholipase A2 (sPLA2) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of this genetic and biochemical heterogeneity has provided important clues to the regulation and function of the individual enzymes. An increasing body of evidence shows that their lipid products, lysophospholipids and free fatty acids, mediate a variety of cellular responses, including plant growth, development, and responses to stress and defense. This review discusses the newly-acquired information on plant sPLA2s including the molecular and biochemical characteristics, and signaling functions of each isoform.     

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.     

Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes

G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.     

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.     

Retinol induces platelet aggregation via activation of phospholipase A2

All-trans-retinol induced aggregation of rabbit platelets, and this effect could be inhibited by a cyclooxygenase inhibitor and a thromboxane A2 (TXA2) receptor antagonist, indicating an essential role for endogenously produced TXA2. We found a two-phase arachidonic acid release in retinol-stimulated platelets. The first phase was induced by the action of retinol alone and not inhibited by TXA2 receptor antagonist. The second phase was induced via synergistic action of retinol and initially generated small amount of TXA2, and was inhibited by the antagonist. Moreover, we discussed that the arachidonic acid release may be mediated by the action of phospholipase A2.     

Structure of genomic DNA for rat platelet phospholipase A2

A genomic DNA for rat platelet phospholipase A2 was isolated by screening a rat genomic library with oligonucleotide probes based on its published amino acid sequence. The rat platelet phospholipase A2 gene had a total length of about 2.5 kb and contained five exons and four introns. The intron-exon structure of the rate gene was similar to that of human non-pancreatic phospholipase A2.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Purification and characterization of rabbit platelet cytosolic phospholipase A2

A phospholipase A2 was purified from rabbit platelet cytosolic fraction to near homogeneity by sequential column chromatographies on heparin-Sepharose, DEAE-Sephacel, butyl-Toyopearl, DEAE-5PW ion-exchange HPLC, and TSK gel G3000SW gel-filtration HPLC. The final preparation with an estimated specific activity of 8630 nmol/min per mg protein, showed a single band with a molecular mass of about 88 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The 88-kDa phospholipase A2 exhibited a fatty acid preference; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10(-7) and 10(-6) M calcium ion, indicating that it could be regulated by less than micromolar concentration of calcium. These characteristics differ from those of platelet secretory 14-kDa phospholipase A2 reported previously. Therefore, this 88-kDa enzyme is a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.     

Identification of a human platelet membrane protein alkylated under conditions inhibitory of phospholipase A2 activity

Platelet membrane sulfhydryls essential for phospholipase A2 activity were alkylated by [3H]N-ethylmaleimide after the non-essential sulfhydryls were cross-linked by azodicarboxylic acid bis(di-methylamide) (DA) or alkylated by N-ethylmaleimide. A 24.5K da protein labeled under phospholipase inhibitory conditions was not labeled under non-inhibitory conditions. The polypeptide, which had neither endogenous nor DA induced disulfides, may be a platelet membrane phospholipase A2 or a lipase regulatory protein.     

Multiple forms of phospholipase A2 in arthritic synovial fluid

Phospholipase A2 (PLA2) has been purified to homogeneity from human arthritic synovial fluid. The activity resolved into multiple peaks by preparative HPLC. The most abundant peak (A) was present in synovial fluid from patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. A second major peak (B) was variable and lower in relative abundance, but was distinguishable from peak A by its stimulated activity in the presence of either 0.5 M Tris or 0.1% sodium deoxycholate (DOC), in addition to its longer HPLC column retention time. Both peaks required Ca2+ and showed optimal activity in DOC/phosphatidylcholine (PC) mixed micelle assays between pH 8.0 and 9.0. Both peaks showed higher activity with PC as substrate than with PI, however peak A exhibited higher activity with PE than PC. Upon preparative SDS-polyacrylamide gel electrophoresis, both peaks of PLA2 activity were resolved as proteins of approximately 14,000 Da. The N-terminal sequence obtained from purified peak A material matched that of a recent similar isolate (Hara et al. (1988) J. Biochem. 104, 326-328).     

Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet

Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 microgram/ml of compound 48/80 slightly stimulated Ca2+-dependent phospholipase A2, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A2 purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved.