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Case Reports
, 118 (6), 2121-31

Inherited Human cPLA(2alpha) Deficiency Is Associated With Impaired Eicosanoid Biosynthesis, Small Intestinal Ulceration, and Platelet Dysfunction

Case Reports

Inherited Human cPLA(2alpha) Deficiency Is Associated With Impaired Eicosanoid Biosynthesis, Small Intestinal Ulceration, and Platelet Dysfunction

David H Adler et al. J Clin Invest.

Erratum in

  • J Clin Invest. 2009 Sep;119(9):2844


Cytosolic phospholipase A2alpha (cPLA2alpha) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA2alpha function has been derived from investigations of the enzyme and from cPLA2alpha-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 B2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 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 cPLA2alpha genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E2, prostaglandin D2, and thromboxane A2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA2.


Figure 1
Figure 1. Arachidonic cascade initiated by cPLA.
P450, cytochrome P450; LOX, lipoxygenase. cPLA reaction is in red, and other enzymatic reactions are in blue.
Figure 2
Figure 2. Gross and histologic pathology.
(A and B) Intraoperative endoscopy shows a well-demarcated ulcer in the proximal ileum with minimal surrounding inflammation. (C) A shallow jejunal ulcer involves the mucosa and submucosa. Acute inflammatory exudate covers the ulcer base (solid arrow). Neutrophils infiltrate the submucosa (dashed arrow). There are no signs of chronicity nor evidence of viral inclusion, inflammatory bowel disease, or vasculitis. Original magnification, ×100. (D) Renal-cell carcinoma, Furman grade II. Original magnification, ×100.
Figure 3
Figure 3. Platelet-derived eicosanoid biosynthesis.
Serum TxB2 was measured by GC/MS, and serum 12-HETE was measured by LC/MS. Normal values for serum eicosanoids were obtained from healthy male volunteers (n = 49 for TxB2; n = 42 for 12-HETE). The lower limits of the reference ranges are represented by the 0.025 quantiles for normal volunteers (brackets represent 0.025 quantiles ± SEM). Mean values are represented by horizontal bars. Individual measurements are plotted for patient (closed circles) and heterozygous family members (open circles).
Figure 4
Figure 4. Ex vivo TxB2 production by washed platelets after the addition of deuterated AA (2 μM).
Endogenous substrate converted by platelet COX-1 was measured as nondeuterated TxB2, whereas exogenous substrate was measured as deuterated TxB2. Both deuterated and nondeuterated TxB2 were measured simultaneously in the same samples after addition of deuterated AA to assess endogenous AA release triggered by exogenous AA. Error bars represent ± SD.
Figure 5
Figure 5. Platelet aggregation and dense granule release.
(A) Representative measurements of platelet aggregation and simultaneous ATP release. Left panel shows aggregation (blue) and ATP release (black) in platelets from a normal control in response to ADP (10 μM). Middle panel shows aggregation (blue and red, performed in duplicate) and ATP release (black and green, in duplicate) in platelets from the proband in response to ADP (10 μM). Right panel shows aggregation (blue and red, in duplicate) and ATP release (black and green, in duplicate) in platelets from the proband in response to AA (500 μM). (B) Optical platelet aggregation in PRP in response to ADP (5 μM) or collagen (2 μg/ml) and ATP release as a measure of platelet degranulation recorded simultaneously during platelet aggregation. Each point represents 1 measurement (different days for the patient and different control volunteers); horizontal bars represent mean.
Figure 6
Figure 6. Platelet PLA2 quantity and activity.
(A) Western blot of platelet lysate from the patient demonstrates cPLA protein of expected molecular weight but in diminished quantity compared with controls. Band intensity was quantified using Quantity One image analysis software (n = 3). Error bars represent ± SD. (B) PLA2 activity in platelet lysate measured by hydrolysis of a radiolabeled substrate, l-3-phosphatidylcholine, 1-stearoyl-2-[1-14C] arachidonyl (14C-SAPC). Points represent mean values for each time point (n = 4; 2 separate experiments performed in duplicate: 2 control volunteers and 2 different collection days for patient). Error bars represent ± SD.
Figure 7
Figure 7. cPLA mutations.
(A) cPLA cDNA sequence chromatograms identifying 3 transitions that encode heterozygous nonsynonymous amino acid substitutions (S111P, R485H, and K651R) in a patient with cPLA deficiency. The patient’s mother was only heterozygous for the S111P alleles, while his sister was heterozygous for both the R485H and K651R alleles. (B) Sequence conservation around each amino acid substitution. Amino acid sequences from human, chicken, rat, dog, mouse, frog, and zebrafish were compared. All 3 identified amino acid changes were highly conserved across species. Sequences were obtained and aligned from the NCBI BLAST database (BLAST 2 sequences (59); blastp 2.2.10;
Figure 8
Figure 8. cPLA tertiary structure and location of amino acid substitutions.
The cPLA structure is depicted as a ribbon diagram with α-helices in red, β-strands as blue arrows, loops in gray, and Ca2+ ions as yellow spheres (center). The locations of described amino acid side-chain substitutions are highlighted in green. Three magnified views highlight each mutation. In the left panel, the Ca2+ binding domain is highlighted and shows the S111 position. The upper panel shows that the catalytic domain contains an active site composed of the catalytic dyad of Ser228 and Asp549. Arg200 is also required for catalytic activity (functionally obligate amino acids are shown with yellow carbon atoms and bonds) (11). Arg485 is in proximity to a cluster of lysine residues (blue) that are essential for interfacial binding of cPLA (12) and for binding PIP2 (13). p.[R485] is located in a cleft in a helical region containing several positively charged residues in the membrane-facing region of the catalytic domain (11), where the side-chain guanido group forms 2 stabilizing hydrogen-bonding interactions within this fold. A “hinged lid,” which prevents exposure of the active site to substrate until interaction with a lipid membrane is shown in purple. The right panel depicts the location of Lys651, the relative position of which has not been determined.
Figure 10
Figure 10. Effects of histidine substitution at residue 485 of cPLA.
Comparison of the wild-type cPLAstructure (left) with a model of the p.[R485H] mutation (right). Backbone trace is shown in gray with the backbone of positively charged positions shown in blue and labeled with a plus sign; the capping helix is highlighted in purple. Oxygen atoms and hydroxy groups are shown in red, nitrogen atoms in blue, and sulfur atoms in yellow. The side-chain carbon atoms of the 485 position are shown in green, and the side-chain atoms of catalytically important residues are highlighted in yellow. Hydrogen bonds are indicated with a dashed black line while distances too long to form a hydrogen-bonding interaction are shown with solid blue lines. Modeling of histidine into position 485 reveals that 2 ideal hydrogen bonds between the side chain of p.[R485] and the backbone carbonyl of p.[T481] and side-chain Sγ of p.[M470] are lost. This mutation is predicted to introduce a destabilizing cavity in the location of the p.[R485] side chain.
Figure 9
Figure 9. Effects of proline substitution at residue 111 of cPLA.
Comparison of the wild-type cPLA structure (left) with a model of the p.[S111P] mutation (right). Backbone carbon atoms are shown in gray, oxygen atoms and hydroxy groups in red, nitrogen atoms in blue, and side-chain carbon atoms of the 111 position in green. Hydrogen-bonding interactions are shown in dashed black lines and sterically unfavorable close distances are shown in solid red lines. A hydrogen-bonding interaction has an ideal distance of 2.7–3.0 υ between oxygen and nitrogen-hydrogen–bond donors and acceptors and an ideal distance of 3.1–3.2 υ for sulfur to nitrogen-hydrogen–bond donors and acceptors. Nonbonded distances smaller than 2.5 υ between nitrogen and oxygen-hydrogen–bonding donors and acceptors are sterically and energetically unfavorable and are generally not observed in nature. Distances longer than 3.5 υ do not contribute favorable energy toward folding stabilization. Modeling of proline in position 111 reveals that 1 ideal hydrogen-bonding interaction of 2.7 υ between the p.[S111] side-chain hydroxyl moiety and the backbone carbonyl group of p.[T108] has been replaced by 3 sterically unfavorable interactions (<2.5 υ). Two of these unfavorable interactions lie between the hydrophobic proline Cγ and Cδ atoms and the p.[T108] carbonyl, while 1 unfavorable interaction is between the proline Cδ and the amide nitrogen of p.[S111]. The p.[S111P] mutation is predicted to cause unfavorable interactions (red lines) in the absence of a structural rearrangement of the protein. The predicted decrease in stability of the adjacent β-strands likely affects the entire Ca2+-binding domain.

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