Lipoprotein-associated phospholipase A2 and its association with cardiovascular outcomes in patients with acute coronary syndromes in the PROVE IT-TIMI 22 (PRavastatin Or atorVastatin Evaluation and Infection Therapy-Thrombolysis In Myocardial Infarction) trial.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with the risk of cardiovascular (CV) events in population-based studies. The prognostic value of Lp-PLA2 in patients with acute coronary syndromes (ACS) has not been established. METHODS AND RESULTS: Plasma levels of Lp-PLA2 activity were measured at baseline (n=3648) and 30 days (n=3265) in patients randomized to atorvastatin 80 mg/d or pravastatin 40 mg/d after ACS in the PROVE IT-TIMI 22 (PRavastatin Or atorVastatin Evaluation and Infection Therapy-Thrombolysis In Myocardial Infarction) trial. The primary end point was death, myocardial infarction, unstable angina, revascularization, or stroke (mean follow-up 24 months). At baseline after ACS, the risk of recurrent CV events was similar across all quintiles of Lp-PLA2 activity (Ptrend=0.88). Overall, mean levels of Lp-PLA2 were lower at 30 days of follow-up than at baseline (35.7 versus 40.9 nmol.min(-1).mL(-1), P<0.001). In particular, treatment with atorvastatin 80 mg/d was associated with a 20% reduction in Lp-PLA2 activity (P<0.001), whereas Lp-PLA2 rose 3.6% with pravastatin 40 mg/d (P<0.001). Patients with 30-day Lp-PLA2 activity in the highest quintile were at significantly increased risk of recurrent CV events compared with those in the lowest quintile (26.4% versus 17.6%, Ptrend=0.002). After adjustment for cardiac risk factors, treatments, achieved low-density lipoprotein (LDL), and C-reactive protein, Lp-PLA2 activity in the highest quintile remained independently associated with a higher risk of recurrent CV events (adjusted hazard ratio 1.33, 95% confidence interval [CI] 1.01 to 1.74). CONCLUSIONS: Lp-PLA2 is not useful for risk stratification when measured early after ACS. At 30 days, Lp-PLA2 activity is significantly lowered with high-dose statin therapy and is associated with an increased risk of CV events independent of C-reactive protein and LDL cholesterol levels.

Natural disruption of group 2 phospholipase A2 gene protects against choline-deficient ethionine-supplemented diet-induced acute pancreatitis and lung injury.

OBJECTIVES: Group 2 phospholipase A2 (PLA2) plays an important role in the pathogenesis of multiple organ failure associated with acute pancreatitis. C57 BL/6J mice are natural group 2 PLA2 knockout mice lacking group 2 PLA2 mRNA. To clarify the role of group 2 PLA2 in the exacerbation of acute pancreatitis, we studied the biologic and histologic alterations in choline-deficient and ethionine-supplemented (CDE) diet-induced pancreatitis in group 2 PLA2-deficient C57 BL/6J mice and compared them with those in wild-type mice. METHODS: Female C57 BL/6J mice weighing 20 to 22 g were fed a CDE diet for 3 days to induce pancreatitis. Female C3H/HEJ mice were used as controls. Mice were killed on days 1, 2, and 3 after the onset of the CDE diet. The severity of pancreatitis was evaluated by survival rate, plasma PLA2 activity, serum amylase level, histologic changes in the pancreas and lung, and myeloperoxidase activity in the lung. RESULTS: The survival rate of C57 BL/6J mice was 100% up to day 3 after the onset of the CDE diet, whereas that of the control mice was 42% on day 3. Plasma PLA2 activity in control mice increased on day 3 but did not increase in C57 BL/6J mice. Serum amylase activity on day 3 in C57 BL/6J mice was 15,480 +/- 3036 SU/dL, which was significantly lower than that in the control mice (43,760 +/- 8657 SU/dL, P < 0.01). Histologic changes in the pancreas of C57 BL/6J mice were markedly milder than in control mice. The degree of alveolar membrane thickening and infiltration of inflammatory cells in the lung of C57 BL/6J mice were overtly less than those of the controls. Myeloperoxidase activity in the lung of C57 BL/6J mice was lower, albeit insignificant, than in C3H/HEJ mice. CONCLUSIONS: Natural disruption of the group 2 PLA2 gene protects against CDE diet-induced acute pancreatitis and associated lung injury. These findings support the view that group 2 PLA2 is one of the factors in the exacerbation of severe acute pancreatitis.

Relative sensitivities of plasma lecithin:cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure.

In order to identify potential atherogenic properties of gas-phase cigarette smoke, we utilized an in vitro exposure model to determine whether the activities of several putative anti-atherogenic enzymes associated with plasma lipoproteins were compromised. Exposure of heparinized human plasma to gas-phase cigarette smoke produced a dose-dependent reduction in the activity of platelet-activating factor acetylhydrolase (PAF-AH). Reductions of nearly 50% in PAF-AH activity were observed following exposure to gas-phase smoke from four cigarettes over an 8-h period. During this time of exposure, lecithin:cholesterol acyltransferase (LCAT) was rendered almost completely inactive (>80%). In contrast, paraoxonase was totally unaffected by cigarette smoke. Supplementation of plasma with 1 mM reduced glutathione was found to protect both PAF-AH and LCAT from cigarette smoke, suggesting that cysteine modifications may have contributed to the inhibition of these two enzymes. To evaluate this possibility, we blocked the free cysteine residues of these enzymes with the reversible thiol-modifying reagent dithiobisnitrobenzoic acid (DTNB). Reversal of the DTNB-cysteine adducts following cigarette smoke exposures revealed that LCAT, but not PAF-AH, was protected. Moreover, high doses (1.0-10 mM) of acrolein and 4-hydroxynonenal, reactive aldehydic species associated with cigarette smoke, completely inhibited plasma LCAT activity, whereas PAF-AH was resistant to such exposures. Taken together, these results indicate a divergence regarding the underlying mechanism of PAF-AH and LCAT inhibition upon exposure to gas-phase cigarette smoke. While LCAT was sensitive to exposure to volatile aldehydic products involving, in part, cysteine and/or active site modifications, the enzyme PAF-AH exhibited an apparent resistance. The latter suggests that the active site of PAF-AH is in a microenvironment that lacks free cysteine residues and/or is shielded from volatile aldehydic combustion products. Based on these results, we propose that cigarette smoke may contribute to atherogenesis by inhibiting the activities of plasma PAF-AH and LCAT, but the nature of this inhibition differs for the enzymes.

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils.

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The equine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA(2) was differently influenced by cations as compared with the human cPLA(2). Second, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa protein, in comparison with human cPLA(2) which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA(2) protein was observed. Thus, the cPLA(2) protein from eosinophils was constitutively phosphorylated, while the cPLA(2) protein from neutrophils was unphosphorylated. In summary, these results demonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPLA(2) protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.

Bradykinin B2-receptor antagonism attenuates fatal cardiocirculatory breakdown induced by severe experimental pancreatitis.

OBJECTIVES: To investigate the impact of the long-acting bradykinin B2 receptor antagonist HOE 140 (Icatibant) on survival time in a model of severe porcine pancreatitis. DESIGN: Randomized, controlled intervention trial. SUBJECTS: Thirty domestic pigs of either gender anesthetized by intravenous application of piritramide, midazolam, and pancuronium and mechanically ventilated. INTERVENTIONS: Pancreatitis was induced by an injection of sodium taurocholate (5%, 1 mL/kg body weight [BW]) and enterokinase (10 U/kg BW). Control animals (group 1, n = 10) underwent the spontaneous course of the disease. In two treatment groups, Icatibant was administered either in a low (100 nmol/kg BW; group 2, n = 10) or in a high dosage (5000 nmol/kg BW; group 3, n = 10). MEASUREMENTS AND MAIN RESULTS: Mean survival time was significantly prolonged by Icatibant (controls, 6.6 hrs; group 2, 9.8 hrs; p = .022; group 3, 10.9 hrs; p = .007). Six hours postinduction, the decline of total peripheral resistance (52% of baseline) and cardiac index (92% of baseline) in controls was significantly improved by Icatibant, both in the low (16% and 44%; p < .05) and high (6% and 45%; p < .05) dosage. The concentrations of free, nonreceptor-bound kinin in plasma 6 hrs postinduction were significantly lower in controls than in groups 2 and 3 animals (111+/-50 vs. 208+/-40 and 237+/-52 fmol/mL, respectively). Six hours postinduction, the pretreatment with Icatibant was associated with significantly higher plasma concentrations of phospholipase A2 (controls, +1194%; group 2, +2000%; group 3, +2285% of baseline values) and interleukin-1 receptor antagonist (controls, 1900+/-800; group 2, 3100+/-800; group 3, 3600+/-800 pg/mL). In contrast, the increase of urinary trypsinogen activation peptides indicating local pancreatic damage (589+/-114 nmol/L in controls) was substantially attenuated by pretreatment with Icatibant (group 2, 467+/-102, NS; 352+/-91 nmol/L in group 3; p = .022 vs. controls). Systemic inflammatory reactions, however, as quantified by C-reactive protein and the extracellularly discharged neutrophil cytosolic inhibitor leukocyte neutral proteinase inhibitor were not influenced by the bradykinin B2-receptor antagonist. CONCLUSIONS: Pretreatment with the bradykinin B2 receptor antagonist Icatibant resulted in prolonged survival time and in delayed impairment of major macrocirculatory and pulmonary variables. Icatibant resulted in elevated concentrations of free, circulating kinin. This was associated with increased concentrations of phospholipase A2 and interleukin-1 receptor antagonist, suggesting that circulating kinins strengthen the activation of some mediator cascades, the association of which with the kinin metabolism requires further experimental clarification. Other variables indicating a systemic inflammatory response (C-reactive protein, leukocyte neutral proteinase inhibitor) remained unaffected by Icatibant. Bradykinin antagonism distinctly ameliorated the local pancreatic damage, indicated by increased urinary concentrations of trypsinogen activation peptides. It is concluded that the kinin metabolism plays an important role in the pathophysiology of systemic complications after severe experimental pancreatitis.

Effects of green tea catechin on phospholipase A2 activity and antithrombus in streptozotocin diabetic rats.

The purpose of this study was to investigate the effects of dietary green tea catechin on phospholipase A2 (PLA2) activity and the antithrombotic reaction of platelets in streptozotocin (STZ)-diabetic rats. Sprague-Dawley male rats weighing 100 +/- 10 g were randomly divided into one normal and three STZ-diabetic groups, which were subdivided into catechin-free group (DM-0C), 0.5% catechin group (DM-0.5C) and 1% catechin group (DM-1C). The activity level of platelet phospholipase A2 was higher in the diabetic groups than in the normal group, while it was lower in DM-0.5C and DM-1C than in DM-0C. The activity of platelet cyclooxygenase in DM-0C was 1.1-fold as high as in the normal group, but was significantly reduced by catechin supplementation. The platelet thromboxane A2 (TXA2) formation became higher in DM-0C as compared to the normal group, but not in DM-0.5C and DM-1C. The synthesis of aortic prostacyclin (PGI2) was lower in DM-0C and DM-0.5C than in the normal group. The PGI2/TXA2 ratio was decreased to 55% in DM-0C, but was restored by catechin supplementation. These results indicate that STZ-diabetic rats are sensitive to platelet aggregation and thrombosis, and that the abnormality can be improved by dietary catechin.

Deficiency of plasma platelet-activating factor acetylhydrolase: roles of blood cells.

Platelet-activating factor (PAF), a potent mediator of inflammation and circulatory shock, is inactivated by the enzyme PAF acetylhydrolase. Plasma PAF acetylhydrolase deficiency occurs even in healthy subjects. We hypothesized that erythrocyte PAF acetylhydrolase could play a supplementary role in this plasma acetylhydrolase deficiency. We examined 1,030 subjects who participated in mass checkups, and assayed plasma and erythrocyte PAF acetylhydrolase. We also investigated the degradation of exogenous PAF by erythrocytes or other blood cells obtained from subjects who exhibited the plasma enzyme deficiency. The incidence of the plasma enzyme deficiency in this general Japanese population was 4.7% (48/1,030). None of the subjects with the deficiency had a history of allergy, circulatory shock, or chronic inflammatory diseases. The mean values for erythrocyte cytosolic PAF acetylhydrolase activity in the normal and deficient subjects were 0.51 +/- 0.15 (SD) and 0.71 +/- 0.28 nkat (nmol/s)/g protein, respectively, and the difference was significant (P < 0.001, Mann-Whitney U-test). The half-life of 10 nmol/l [3H]PAF in plasma from normal subjects was about 5 min, and the half-life in whole blood or erythrocyte suspension in autologous plasma was almost the same as that in plasma. In plasma from deficient subjects, unchanged PAF virtually remained and the degradation in whole blood or erythrocyte suspension was a little faster than in plasma. We conclude that erythrocytes contribute only little to PAF metabolism in normal blood but they account for almost all of the slow PAF degradation in blood from subjects deficient in plasma PAF acetylhydrolase.

Group II phospholipase A2 in synovial fluid and serum in acute arthritis.

Increased catalytic activity of synovial-type (group II) phospholipase A2 (syn-PLA2), has been associated with cartilage erosions in rheumatoid arthritis and osteoarthritis. The catalytic activity of phospholipase A2 and the concentration of syn-PLA2 were measured in a prospective study in synovial fluid (SF) samples from 66 patients with acute knee joint effusion. The median (range) of the concentration of syn-PLA2 in SF was 210 micrograms/l (80-1480 micrograms/l) in culture-positive septic arthritis, 460 micrograms/l (270-1040 micrograms/l) in reactive arthritis, 780 micrograms/l (120-2710 micrograms/l) in osteoarthritis and 230 micrograms/l (80-1400 micrograms/l) in traumatic joint effusions. High concentrations of syn-PLA2 are found also in SF of patients with arthritides not expected to lead to permanent destruction of cartilage.

Lipolytic enzyme and phospholipid level changes in intraoperative salvaged blood.

Autotransfusion is becoming increasingly popular, mainly because it eliminates the risk of disease transmission. One of the techniques available is intra-operative blood salvage and retransfusion with or without washing of the collected blood. The blood collected during this process is subjected to a variety of chemical and physical insults which can alter the normal composition of the plasma by activating plasma and cellular homeostatic mechanisms. In this study, we measured the plasma levels of total phospholipids, lysolecithin and non-esterified fatty acids, and the lipolytic enzymes phospholipase A2 (PLA2) and lipase in the salvaged blood before and after washing. In the unwashed salvaged blood the mean levels of PLA2, non-esterified fatty acids and lysophospholipids increased by 144, 96 and 149%, respectively, while those of total phospholipids and lipase did not change to any extent. All these substances were reduced to well below the patients circulating plasma levels by washing the collected blood. The changes indicate that the lipid profile of salvaged blood is significantly altered and that potentially dangerous substances such as PLA2 and its metabolites, lysolecithin and non-esterified fatty acids, are present in increased amounts. Washing the blood is recommended prior to reinfusion.

Short term vitamin E supplementation has no effect on platelet function, plasma phospholipase A2 and lyso-PAF in male volunteers.

1. Based largely upon in vitro studies, vitamin E has been reported to inhibit phospholipase A2 activity, to alter phospholipid metabolism and reduce platelet aggregation. 2. The effect of dietary supplementation with D-alpha-tocopherol (1500 iu/day for 14 days) was studied in nine males, 41-63 years old, comparing active treatment with a preceding placebo period. 3. Despite an increase from 2.6 +/- 0.8 (s.d.) x 10(-5) mol/L to 6.0 +/- 1.8 10(-5) mol/L in plasma vitamin E there were no significant changes in the aggregation of diluted whole blood or platelet rich plasma to adenosine diphosphate (ADP) or collagen, in plasma phospholipase A2 activity or plasma lyso-platelet-activating factor (lyso-PAF) (bioassay after in vitro acetylation to PAF). 4. High dose vitamin E dietary supplementation had no effect on these phospholipid and platelet parameters.

[Alpha-tocopherol protection of erythrocytes from hemolysis induced by thermal injury]. / Zashchita al'fa-tokoferolom éritrotsitov ot gemoliza, indutsirovannogo termicheskoi travmoi.

It is shown that skin burn is accompanied by activation of lipid peroxidation (accumulation of TBA-reactive substances and of fluorescent end-products) in the blood of experimental animals. The decrease in red blood cell membrane stability was demonstrated exerting as increase in the rate of spontaneous hemolysis, content of extraerythrocyte++ haemoglobin and increased sensitivity to exogenous oleic acid. It is estimated that alpha-tocopherol possesses protective stabilizing effect on red blood cell membrane. This stabilizing action is observed when alpha-tocopherol was injected both before the skin burn and immediately after it. It is concluded that two different mechanisms are responsible for stabilizing effect of tocopherol, namely: 1) antiradical, realized via inhibition of lipid peroxidation, and 2) non-antioxidant, caused by interaction of tocopherol with phospholipid hydrolysis products by phospholipases A2 (free fatty acids and lysophospholipids).

Rat platelet arachidonate metabolism in the presence of Ca2+, Sr2+ and Ba2+: studies using intact platelets and semi-purified phospholipase A2.

To document further the involvement of external Ca2+ in the platelet-induced activation process, we have studied the arachidonate metabolism of intact washed rat platelets in the presence of different concentrations of Ca2+, Sr2+ or Ba2+. The thrombin-induced mobilization of radiolabeled arachidonate preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products. Results indicate that upon thrombin stimulation (0.2 U/ml), the release of endogenous arachidonate and the formation of its metabolites are reduced by 50-90% only by omission of Ca2+ as compared to 1 mM Ca2+ in the suspending medium. At higher Ca2+ concentrations (5 mM), the arachidonate mobilization and metabolite formation are inhibited and the data are thus close to those obtained in the absence of Ca2+. In the presence of Sr2+ or Ba2+, the results indicate that these cations can substitute for Ca2+. As for Ca2+, an optimum concentration is found for Sr2+ and Ba2+ (3-5 mM), and higher concentrations inhibit the metabolism of arachidonic acid. As the above data might be compatible with the possible entry of Sr2+ and Ba2+ into platelets upon stimulation, we also studied the activity of a semi-purified preparation of phospholipase A2 from rat platelets. This activity was assayed (pH 9.2) using heat-denatured [3H]arachidonate-prelabeled phospholipids as substrate. The results show that this phospholipase A2 activity was strongly Ca2+-dependent. In addition, we found that unlike Mg2+, Sr2+ and Ba2+ are able to greatly enhance this activity. Relative efficiency (Vmax) was in the order Ca2+ greater than Sr2+ greater than Ba2+. Taken together, these findings suggest that external Ca2+ may play a major role in the regulation of rat platelet activity. Our interpretation is in line with the view that Sr2+ or Ba2+ could enter the platelet through a mechanism common to Ca2+ (a Ca2+ channel). Although direct evidence is awaited from the results of further studies which are in progress, it can reasonably be considered that Sr2+ or Ba2+ might cause platelet-induced activation mimicking a rise in the cytosolic Ca2+ and subsequent activation of Ca2+-dependent enzymes.

Phospholipase A2 inhibitors and their possible clinical use in the treatment of acute pancreatitis.

A laboratory method was established for measurement of phospholipase A2 activity in buffer and serum. A series of different phospholipase A2 inhibitors was tested. The most effective inhibitors were Ca2+ chelating compounds like EDTA, DTPA, EGTA, and phytic acid. The calcium salt of EDTA also has some inhibitory effect. Serum phospholipase A2 activity in normal healthy control patients was measured. The activity in 27 patients with acute pancreatitis was tested. The activity was abnormally high in five patients. This activity was in vitro inhibited by EDTA and partly by CaNa2EDTA. The clinical picture of these patients did not differ from that of phospholipase-A2-negative patients. Six patients with acute pancreatitis were treated by intravenous infusion of CaNA2EDTA. Two of them had haemorrhagic pancreatitis and two were suspected of having early haemorrhagic pancreatitis. During the CaNa2EDTA infusion serum amylase and phospholipase A2 activities decreased. All patients recovered. No harmful side effects were noticed.