Shigeru Takamatsu

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2.

The human hepatoma cell line, HepG2, secreted an activity that degrades platelet-activating factor (PAF) by the hydrolysis of the sn-2 acetyl group. This activity was Ca++ independent, inhibited by diisopropylfluorophosphate but not by p-bromophenacyl bromide, and resistant to treatment with trypsin or pronase. Separation of HepG2-conditioned medium by gel filtration disclosed that the activity was associated with lipoproteins. An antiserum against PAF acetylhydrolase immunoprecipitated this activity. It was not recognized by an antibody against lecithin:cholesterol acyltransferase (LCAT), which also is secreted by HepG2 cells. Therefore the phospholipase A2 activity of LCAT was excluded as a source of the observed activity. PAF added to the culture medium stimulated the secretion of the PAF-degrading activity by HepG2 cells, while lyso-PAF was inactive. Maximal stimulation was observed with 5 ng/ml PAF, which induced a fivefold increase. The presence of 5 ng/ml PAF, enhanced the secretion of [35S]methionine-labeled PAF acetylhydrolase and cycloheximide inhibited both the basal and PAF-stimulated secretion of the labeled enzyme. We conclude that HepG2 cells produce PAF acetylhydrolase. The liver may be a major source of plasma PAF acetylhydrolase, and PAF may induce the production of its inactivating enzyme by the liver.

Platelet-activating factor acetylhydrolase in plasma lipoproteins from patients with ischemic stroke.

Background and Purpose Platelet-activating factor is a potent bioactive phospholipid and may play an important role in occlusive vascular diseases. To assess the inactivation of this autacoid in plasma, we measured platelet-activating factor acetylhydrolase activity in plasma low density and high density lipoproteins from patients with ischemic stroke. Methods Low density and high density lipoproteins were separated by ultracentrifugation from plasma of 33 patients with cerebral thrombosis and 31 age-matched healthy control subjects, and plateletactivating factor acetylhydrolase activity in each fraction was assayed. Results The average values of platelet-activating factor acetylhydrolase activity in low density lipoprotein from patients and control subjects were 41±18 and 29±17 nmol/ml per minute, respectively, and the difference was statistically significant (p<0.01, U test). There was no difference in activity in high density lipoprotein between the two groups (16±11 versus 14±9 nmol/ml per minute, respectively). Conclusions The increased plasma platelet-activating factor acetylhydrolase activity in stroke patients is primarily attributable to the increased binding to low density lipoprotein, and this increase may be an adaptation to the augmented generation of platelet-activating factor in ischemic stroke.

Increased activity of the platelet-activating factor acetylhydrolase in plasma low density lipoprotein from patients with essential hypertension

We have measured activity of platelet-activating factor (PAF) acetylhydrolase, an enzyme that specifically inactivates PAF, in plasma from patients with essential hypertension and healthy controls. The average activities in 34 patients and 22 controls were 113 +/- 60 and 79 +/- 32 nmol/ml/min, respectively, and the difference was significant (p less than 0.05). Approximately three fourths of the total plasma activity was recovered in LDL, with the remainder in HDL; and there was a significant difference in the activity associated with the LDL between patients and controls. The relative distribution of the activity among lipoproteins was almost equal in the two groups, and there was no difference in plasma lipids or apoproteins between them. In patients there was a tendency for plasma PAF acetylhydrolase activity to increase with the length of the history of hypertension. Further studies are needed to distinguish between a number of reasons for increased levels of plasma PAF acetylhydrolase in essential hypertension.

Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from patients with ischemic cerebrovascular disease

Platelet-activating factor (PAF) is metabolized by a specific enzyme, PAF acetylhydrolase, which may play an important role in the manifestation of the biological activities of PAF in vivo. The activity of PAF acetylhydrolase in plasma of patients with ischemic stroke was higher than that in healthy controls. The incidence of irreversible platelet aggregation in response to PAF, as well as to ADP, was found to be higher in patients than in controls. The patients whose platelets responded with irreversible aggregation to PAF displayed a higher activity of plasma PAF acetylhydrolase than those with only reversible aggregation. In controls, PAF acetylhydrolase activity correlated positively, although weakly, with LDL-cholesterol, which may reflect the major role of LDL in carrying this enzyme. However, since there was no significant difference in plasma levels of lipids and apoproteins between patients and controls (except for apo B) and there was no significant relationship between the enzyme activity and the levels of other lipids and apoproteins, it is unlikely that increased plasma level of PAF acetylhydrolase activity in stroke patients is accounted for by an abnormality of lipoprotein metabolism. Platelet hyperfunction may be associated with augmented generation of PAF, which, in turn, may bring about the induction of the inactivating enzyme, PAF acetylhydrolase.

Effect of cigarette smoking on the levels of platelet-activating factor-like lipid(s) in plasma lipoproteins

The effect of cigarette smoking on the levels of platelet-activating factor-like lipid(s) (PAF-LL) in plasma lipoproteins was studied. The subjects were 10 healthy male non-smokers (24 +/- 1.4 years old) and 13 healthy male habitual smokers (23 +/- 1.3 years old). Fasting venous blood was obtained and basal levels of PAF-LL in plasma lipoproteins were estimated. The acute effect of cigarette smoking was also studied in smokers. Plasma lipoproteins were separated by ultracentrifugation. Lipids were extracted and separated by thin-layer chromatography. The fraction with the same migration as authentic PAF was recovered and was shown to cause aggregation of human polymorphonuclear neutrophils. This activity was identified as PAF-LL because it was inactivated by phospholipase A2 and was blocked by CV-3988, an antagonist of the PAF receptor. PAF-LL was detected in LDL and HDL, but not in VLDL or in lipoprotein-deficient plasma. The levels of PAF-LL in LDL in non-smokers, and in smokers before and after smoking were 13 +/- 7.5, 16 +/- 14.9 and 190 +/- 179.0 pg/ml, and those in HDL were 12 +/- 5.2, 40 +/- 40.0 and 235 +/- 205.1 pg/ml, respectively. The values in both LDL and HDL in smokers increased significantly after smoking (P less than 0.05). After 30 min, the levels had returned almost to the pre-smoking levels. We conclude that cigarette smoking induces an increase in the levels of PAF or closely related lipid(s) in LDL and HDL, which may be related to smoking-induced atherosclerosis.

Increased levels of blood platelet-activating factor (PAF) and PAF-like lipids in patients with ischemic stroke.

Levels of platelet‐activating factor (PAF) in blood from patients with ischemic stroke were determined by radioimmunoassay (RIA). Using 2 ml of blood as a starting material, PAF was detected in 11 out of 17 stroke patients and 3 of 25 age‐matched healthy controls. This implies that blood level of PAF is higher in stroke patients than in controls. Plasma levels of PAF‐like lipid(s) (PAF‐LL) were also estimated in the same subjects by a bioassay based on aggregation of human polymorphonuclear neutrophils. PAF‐LL was detected in plasma samples of all subjects and the average values in patients and controls were 294 ± 211 pg/ml and 140 ± 122 pg/ml, respectively. There was a statistically significant difference between these two values (p < 0.01). Separation of plasma lipids by HPLC gave a single peak in bioassay, which had the same elution volume as authentic PAF. When each fraction was subjected to RIA, the fractions corresponded to phosphatidylcholine (PC) or lysoPC also showed the immunoreactivity, however, the purification procedure using an octadecylsilica gel cartridge eliminated such cross‐reacting compounds. We conclude that blood PAF is higher in patients with ischemic stroke than in healthy subjects. Besides, there may be bioactive phospholipid molecules other than PAF, which level in plasma is also higher in stroke patients.

Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from healthy habitual cigarette smokers

SummaryThe activity of platelet-activating factor acetylhydrolase, a specific metabolizing enzyme of platelet-activating factor (PAF), in plasma of 39 male subjects was determined radiochemically by the method of Stafforini et al., (1987) J Biol Chem 262: 4223–4230, to clarify to what extent PAF affects atherosclerotic disorders induced by habitual smoking. The subjects examined included 18 habitual smokers (mean age: 62±10.6 years) and 21 age-matched nonsmokers. Plasma PAF acetylhydrolase activity was higher in the smokers than in the nonsmokers. There was no difference between smokers and nonsmokers in platelet aggregability in response to PAF or ADP. A higher apoprotein B/apoprotein A-I ratio in smokers as compared to nonsmokers was the only manifestation of abnormal lipoprotein metabolism in the former group. In the smokers, plasma PAF acetylhydrolase activity was directly proportional to total cholesterol, LDL-cholesterol, triglyceride, apoprotein B, LDL-cholesterol/HDL-cholesterol or apoprotein B/apoprotein A-I, and inversely proportional to HDL-cholesterol or apoprotein A-I. The results obtained suggest that alterations in PAF acetylhydrolase levels result from a slightly abnormal lipoprotein metabolism. Determination of plasma PAF acetylhydrolase activity is useful to study the role of PAF in atherosclerotic changes induced by smoking.

Platelet-activating factor acetylhydrolase in red cell membranes. Does decreased activity impair erythrocyte deformability in ischemic stroke patients?

Background and Purpose Platelet-activating factor acetylhydrolase hydrolyzes platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). It also hydrolyzes oxidized derivatives of phosphatidylycholine that have a short-chain acyl residue at the sn-2 position. This enzyme may act mainly in the degradation of oxidized phospholipids and may play a role in maintaining erythrocyte deformability. Therefore, we assessed the activity of red cell membrane platelet-activating factor acetylhydrolase in patients with ischemic stroke and studied the relation of the enzyme activity to red cell deformability. Methods Enzyme activity was measured in the detergent extract of red cell membranes from 38 patients with cerebral thrombosis and 38 age-matched healthy volunteers. Red cell filterability, an index of red cell deformability, was also measured. Results The enzyme activity in patients and control subjects was 100±74 and 148±128 nmol/g protein per minute (2.68 ±.11 and 3.79 ± 2.46 pmol/109 cells per minute) (mean ± SD), respectively, and the difference was significant (p<0.05 by the Mann-Whitney U test, two-sided test). Enzyme activity was correlated positively with red cell filterability in the patients (n=20, r=0.565, p<0.01). Conclusions Red blood cells from stroke patients have lower levels of platelet-activating factor acetylhydrolase activity when compared with those from healthy subjects. This may result in the accumulation of oxidized lipids in the cell membrane and lead to impaired red cell deformability in patients with cerebral thrombosis.

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.