A mutation in plasma platelet-activating factor acetylhydrolase (Val279-->Phe) is a genetic risk factor for stroke

BACKGROUND AND PURPOSE: Platelet-activating factor (PAF) is a phospholipid with multiple actions that include thrombosis and inflammation. It is inactivated by a plasma enzyme, PAF acetylhydrolase. Deficiency of this enzyme in plasma is caused by a missense mutation in the gene (Val279-->Phe). We have studied a possible association of this mutation with the risk of stroke. SUBJECTS AND METHODS: We studied 120 consecutive patients with cerebral thrombosis. The control group consisted of 134 patients matched for age and sex with minor complaints but without stroke. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF acetylhydrolase activity was determined by the method of Stafforini et al. RESULTS: The prevalence of the mutant gene was 43.4% in stroke patients (39.2% heterozygotes and 4.2% homozygotes), which was significantly higher than the 25.4% in control subjects (22.4% heterozygotes and 3.0% homozygotes) (chi 2 = 9.22, P < .01). The prevalence was slightly higher in stroke patients without hypertension than those with hypertension, but the difference was not significant. The patients with family histories of stroke had a slightly higher but not a significant prevalence of the mutant gene as compared with those without family histories of stroke. Plasma PAF acetylhydrolase activity was higher in patients than in control subjects, in normal subjects, or patients with a heterozygous genotype. CONCLUSIONS: These results suggest that plasma PAF acetylhydrolase deficiency may be a risk factor for stroke. This may explain the relatively high prevalence of stroke in Japan, as the mutation is more common among Japanese than Caucasians.     

Serum paraoxonase and platelet-activating factor acetylhydrolase in chronic renal failure

PURPOSE: Since clinically apparent varicoceles may affect testicular volume and sperm production, early repair has been advocated. However, repair of the pediatric varicocele with conventional nonmagnified techniques may result in persistence of the varicocele after up to 16% of these procedures. Also testicular artery injury and postoperative hydrocele formation can occur after nonmagnified repair. The microsurgical technique has been successfully completed in a large series of adults with a dramatic reduction in complication and recurrence rates. We report our experience with the microsurgical technique in boys. MATERIALS AND METHODS: A total of 30 boys (average age 15.9 years) underwent 42 microsurgical varicocelectomies (12 bilateral). All patients had a large left varicocele. Indications for repair included testicular atrophy (size difference between testicles of greater than 2 ml.) in 20 boys, pain in 5 and a large varicocele without pain or testicular atrophy in 5. Six boys were referred following failure of conventional nonmicrosurgical techniques. All boys were examined no sooner than 1 month postoperatively (mean followup 12). RESULTS: Preoperative volume of the affected testis averaged 13.0 ml., and an average size discrepancy between testicles of 2.8 ml. was noted before unilateral varicocelectomy. No cases of persistent or recurrent varicoceles were detected, and 1 postoperative hydrocele resolved spontaneously. After unilateral varicocelectomy the treated testes grew an average of 50.1%, while the contralateral testes grew only 23%. Overall, 89% of patients with testicular atrophy demonstrated reversal of testicular growth retardation after unilateral varicocelectomy. In contrast, both testes showed similar growth rates after bilateral varicocelectomy (45% left testis, 39% right testis). CONCLUSIONS: The meticulous dissection necessary to preserve arterial and lymphatic supply, and to ligate all spermatic veins in the pediatric patient is readily accomplished using a microsurgical approach, and results in low recurrence and complication rates. Rapid catch-up growth of the affected testis after microsurgical varicocelectomy suggests that intervention during adolescence is effective and warranted.     

The role of recombinant platelet-activating factor acetylhydrolase in a neonatal rat model of necrotizing enterocolitis

Previous studies have shown that the endogenous inflammatory mediator platelet-activating factor (PAF) plays an important role in the pathophysiology of neonatal necrotizing enterocolitis (NEC). This study was designed to investigate the role of the PAF-degrading enzyme acetylhydrolase (PAF-AH) in a neonatal rat model of NEC. To study the absorption, localization, and activity of human recombinant PAF-AH (rPAF-AH), newborn rats were treated with enteral rPAF-AH, and plasma and intestines were sampled at 8 and 24 h for determination of PAF-AH enzyme activity and rPAF-AH concentration using a specific enzyme-linked immunoassay. To study the effect of rPAF-AH on neonatal NEC, rats were treated with rPAF-AH via the enteral route every 3 h, and then subjected to formula feeding and asphyxia per an established neonatal rat protocol for NEC. Pretreatment with enteral rPAF-AH significantly reduced the incidence of NEC compared with controls (6/26 versus 19/26, p < 0.001). We found that enteral rPAF-AH administration resulted in significant intestinal PAF-AH activity but no circulating PAF-AH activity despite immunohistochemical localization of the administered rPAF-AH to the intestinal epithelial cells. These findings suggest that rPAF-AH is functional and stable in the gut of neonatal rats. We conclude that enteral administration of rPAF-AH remains locally active and reduces the incidence of NEC in our experimental animal model.     

Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.     

Effect of recombinant platelet-activating factor acetylhydrolase on two models of experimental acute pancreatitis

BACKGROUND & AIMS: Recent reports suggest that platelet-activating factor (PAF) plays a role in pancreatitis and pancreatitis-associated lung injury. In this study, the effects on these processes of termination of PAF action by recombinant PAF-acetylhydrolase (rPAF-AH) were investigated. METHODS: Rats were given rPAF-AH and then infused with a supramaximally stimulating dose of cerulein to induce mild pancreatitis. Opossums underwent biliopancreatic duct ligation to induce severe pancreatitis, and rPAF-AH administration was begun 2 days later. RESULTS: In mild, secretagogue-induced pancreatitis, rPAF-AH given before the cerulein reduced hyperamylasemia, acinar cell vacuolization, and pancreatic inflammation but did not alter pancreatic edema or pulmonary microvascular permeability. In severe, biliopancreatic duct ligation-induced pancreatitis, rPAF-AH delayed and reduced the extent of inflammation and acinar cell injury/necrosis and completely prevented lung injury even though the rPAF-AH administration was begun after the onset of pancreatitis. CONCLUSIONS: PAF plays an important role in the regulation of pancreatic injury but not pancreatic edema or increased pulmonary microvascular permeability in mild, secretagogue-induced pancreatitis. PAF plays a critical role in the regulation of progression of pancreatic injury and mediation of pancreatitis-associated lung injury in severe biliary pancreatitis. Amelioration of pancreatitis and prevention of pancreatitis-associated lung injury can be achieved with rPAF-AH even if treatment is begun after pancreatitis is established.     

Loss of activity of plasma platelet-activating factor acetylhydrolase due to a novel Gln281-->Arg mutation

The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994-->T mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (<10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of <10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001-->G missense mutation, resulting in a Gln281-->Arg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281-->Arg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity.     

Study of platelet-activating factor acetylhydrolase in the perioperative period of patients undergoing cardiac surgery

This study was conducted to determine whether brief, intermittent exposure to hypoxia with little change in nutrient intake would affect fetal growth. Pregnant rats were exposed to 1 or 2 h of hypoxia (FiO2 = 0.09-0.095) from days 15 to 19 of gestation. Exposure to 1 h of hypoxia decreased fetal body weight and length, liver weight and increased the brain/liver weight ratio (p < 0.05) as compared to controls. Two hours of hypoxia decreased fetal body weight and length, and heart, lung, kidney, gut, brain and liver weights (p < 0.01), but did not affect the brain/liver weight ratio. Two hours of hypoxia decreased maternal food intake and weight gain (p < 0.05), but fetal growth was not significantly altered in pair-fed controls. These data demonstrate that brief, intermittent periods of intrauterine hypoxia have significant effects on fetal growth.     

Effect of 17 beta-estradiol on secretion of platelet-activating factor acetylhydrolase by HepG2 cells

Estrogen has been shown to decrease plasma platelet-activating factor (PAF) acetylhydrolase activity, but the precise mechanisms are not known. We examined the effect of estradiol on the secretion of PAF acetylhydrolase by HepG2 cells. In our previous study, we demonstrated the production of this enzyme by HepG2 cells, which we used as an experimental model of normal hepatocytes. 17 beta-Estradiol mildly but consistently inhibited the secretion of PAF acetylhydrolase by HepG2 cells in a concentration-dependent manner. Under basal conditions, HepG2 cells secreted 42.3 pmol/mg cell protein/min PAF acetylhydrolase in 24 hours (mean of 8 dishes), and the presence of 10(-7) mol/L 17 beta-estradiol decreased the secretion to 77% +/- 10.3% of control values (mean +/- SD, n = 8, P < .02). 17 beta-Estradiol treatment affected neither the secretion of apolipoprotein (apo) A-I nor cell-associated PAF acetylhydrolase activity. Electrophoretic separation of [35S]methionine-labeled PAF acetylhydrolase revealed a single band whose molecular weight was approximately 43,000 d. We conclude that estrogen decreases the secretion of PAF acetylhydrolase by the liver, and it may explain, at least in part, the effect of estrogen on plasma PAF acetylhydrolase.     

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.     

Purification and characterization of platelet-activating factor acetylhydrolase from peritoneal fluid obtained from guinea pigs after endotoxin shock

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetyl-hydrolase is an enzyme that hydrolyzes the acetyl ester of PAF. We purified this enzyme, which accumulated in the peritoneal cavity during endotoxin shock, by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl, heparin-Sepharose, Con A-Sepharose, chelating-Sepharose, and MonoQ column chromatographies. We identified a monomeric polypeptide with a molecular weight of approximately 63 kDa on SDS-PAGE. This molecular weight differs from those of previously described acetylhydrolases. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. In addition, the enzyme activity was not inhibited by either pBPB or quinacrine. Accordingly, this enzyme is distinct from phospholipase A2. In addition, this enzyme also hydrolyzed some oxidatively fragmented phospholipids with PAF-like biological activities such as 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-succinoyl-sn-glycero-3-phosphocholine.     

Administration of a receptor antagonist for platelet-activating factor during equine endotoxaemia

Platelet-activating factor (PAF) is an important mediator of endotoxaemia and various PAF receptor antagonists prevent many of the adverse effects of experimental endotoxaemia in laboratory animals. In this study a specific PAF receptor antagonist was used to investigate the role of PAF in equine endotoxaemia. At an interval of not greater than 10 days, 6 horses were each challenged with endotoxin and endotoxin with concurrent administration of SRI 63-441, a PAF receptor antagonist. The order of the treatments was randomised. Clinical signs, serum biochemical and coagulation profiles, and platelet aggregation in vitro were monitored in all horses for 24 h after treatment. Challenge with endotoxin increased maximal platelet aggregation induced by PAF. This response was blocked by administration of SRI 63-441 concurrently with endotoxin. No changes in percentage maximal platelet aggregation to ADP or collagen were noted after administration of endotoxin. The PAF receptor antagonist delayed the onset of fever, tachycardia, leucopenia and lactic acidaemia. Lack of more profound beneficial alterations of the horses' responses to endotoxin may have been due to the low dose of endotoxin administered in this model or to only partial effectiveness of SRI 63-441 in blocking the effects of endotoxin-induced PAF.     

The vascular distribution of the platelet-activating factor receptor

Although the platelet-activating factor (PAF) is the most active inflammatory mediator known to date, little is known about its effects on the vascular endothelium and about the cellular and subcellular distribution of its receptor, already identified as a membrane protein of approximately 39 kDa. To better understand its functions we decided: i) to study PAF effects on a model microvascular bed (the rat cremaster), ii) to raise monoclonal antibodies against synthetic peptides reproducing short segments (14 and 16 amino acids) at the N and C terminal parts of PAF-receptor (PAF-R), iii) to determine the distribution of PAF-R on a number of microvascular beds. Topical application of the PAF on the cremaster led promptly to: i) opening of the venular and capillary endothelial junctions; ii) fenestration of the endothelium and iii) swelling, clustering and fusion of endothelial plasmalemmal vesicles. With the anti-N terminal antibody, we localized PAF-R by immunofluorescence on semithin frozen sections of lung, heart, diaphragm, kidney, and brain specimens. With the exception of brain, the signal was restricted primarily to the vascular endothelium. Using immunogold procedures, we localized the PAF-R in small clusters on endothelial surfaces and found it associated preferentially with the plasmalemma proper, rather than to any differentiated microdomain. A morphometric analysis revealed a greater signal density at the level of the venular endothelium than at the level of the endothelium of any other segment of the microvasculature. With the same antibody, we immunoprecipitated PAF-R from whole homogenates of the same tissues. The results obtained were in general agreement with the immunofluorescence tests.     

Discovery and evaluation of a series of 3-acylindole imidazopyridine platelet-activating factor antagonists

Studies conducted with the goal of discovering a second-generation platelet-activating factor (PAF) antagonist have identified a novel class of potent and orally active antagonists which have high aqueous solubility and long duration of action in animal models. The compounds arose from the combination of the lipophilic indole portion of Abbott's first-generation PAF antagonist ABT-299 (2) with the methylimidazopyridine heterocycle moiety of British Biotechnology's BB-882 (1) and possess the positive attributes of both of these clinical candidates. Structure-activity relationship (SAR) studies indicated that modification of the indole and benzoyl spacer of lead compound 7b gave analogues that were more potent, longer-lived, and bioavailable and resulted in the identification of 1-(N, N-dimethylcarbamoyl)-4-ethynyl-3-[3-fluoro-4-[(1H-2-methylimidazo[4,5-c] pyrid-1-yl)methyl]benzoyl]indole hydrochloride (ABT-491, 22 m.HCl) which has been evaluated extensively and is currently in clinical development.