Cytosolic phospholipase A 2α (cPLA 2α ) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA 2α function has been derived from investigations of the enzyme and from cPLA 2α -null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane B 2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B 4 released from calcium ionophore-activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patient's cPLA 2α genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA 2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E 2 , prostaglandin D 2 , and thromboxane A 2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA 2 .

Meta-analyses have demonstrated that low dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the anti-platelet action of low dose aspirin likely contributes to its anti-metastatic effect. Cycloxooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low dose aspirin also inhibits PGE2 biosynthesis. We show that low dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (p <0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells up-regulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet-tumor cell aggregates. In conclusion, low dose aspirin has a significant effect on extraplatelet cyclooxygenase, and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells.

Purpose-Small intestinal ulcers are frequent complications of therapy with non-steroidal antiinflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine. Methods-Eicosanoids and metabolites were measured by isotope-dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR. NIH Public AccessResults-We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45 year old male who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B 2 (TxB 2 ) production was 4.6% of normal controls (p<0.006) and serum 12-HETE was 1.3% of controls (p<0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB 4 biosynthesis by ionophore activated leukocytes was only 3% of controls and urinary LTE 4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A 2 -α (cPLA 2 -α) which regulates cyclooxygenase and lipoxygenase mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA 2 -α cDNA demonstrated 2 heterozygous non-synonymous single base pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known SNP: Lys651Arg (K651R).Conclusion-Characterization of this cPLA 2 -α deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity, and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers.

Group IVA cytosolic phospholipase A 2 (cPLA 2 α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA 2 α activity, which was associated with mutations in both alleles coding for the enzyme. In this paper we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity and that the known polymorphism K651R was associated with slightly higher activity than wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA 2 α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the mutation S111P hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity leads to inactivation of the enzyme. Interestingly, our results show that the common polymorphism K651R confers slightly higher activity to the enzyme suggesting a role of this residue in favoring a catalytically active conformation of cPLA 2 α. Our results define how the mutations negatively influence cPLA 2 α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.Group IVA cytosolic phospholipase A 2 (cPLA 2 α) is a widely expressed enzyme in mammalian cells that catalyzes the hydrolysis of phospholipids in the sn-2 position to release arachidonic acid (AA). It is stimulated in response to diverse factors and represents CORRESPONDING AUTHOR FOOTNOTE: Olivier Boutaud, Ph.D., 536 RRB, 2200 Pierce Avenue, Nashville, Tel: (615) Fax: (615) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the first step of metabolic cascades leading to generation of important lipid mediators such as prostaglandins and leukotrienes. These eicosanoids mediate physiological and pathophysiological processes, making it important to understand the factors regulating cPLA 2 α activity. Two knock-out mouse models provided important information concerning the physiological function of cPLA 2 α (1-3).Recently we described a patient with globally decreased eicosanoid production, intestinal ulcers, and platelet dysfunction (4). We showed that these conditions were associated with loss-of-function mutations in both alleles coding for cPLA 2 α. Release of thromboxane (TxA 2 ) and 12-hydroxyeicosatetraenoic acid (12-HETE) by the patient's platelets was reduced by more than 95% compared to control individuals, suggesting that cPLA 2 α is the mai...

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