Lipoproteins Alter the Catalytic Behavior of the Platelet-Activating

Home , Abetalipoproteinemia

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2393-2397, April 1989 Medical Sciences Lipoproteins alter the catalytic behavior of the platelet-activating factor acetylhydrolase in human plasma (inflammation/thrombosis/phospholipases/low density lipoprotein) DIANA M. STAFFORINI, M. ERIC CARTER, GuY A. ZIMMERMAN, THOMAS M. MCINTYRE, AND STEPHEN M. PREscorr Nora Eccles Harrison Cardiovascular Research and Training Institute and Departments of Internal Medicine and Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112 Communicated by Philip W. Majerus, December 15, 1988

ABSTRACT Platelet-activating factor (PAF) has been im- between the two particles (6). This distribution is interesting plicated as a mediator of inflammation, allergy, shock, and because the two particles do not have a common metabolic thrombosis. A specific degradative enzyme, PAF acetyihydro- pathway, but both are involved in cholesterol homeostasis lase (EC 3.1.1.47), is found in plasma and could regulate the (reviewed in ref. 10). concentration of PAF in blood. In plasma, 70% of the PAF We found (6, 9) that the PAF acetylhydrolase activities in acetylhydrolase is found with low density lipoprotein (LDL), LDL and HDL appear to be the same protein and have and the remainder is in high density lipoprotein (HDL). In identical behavior in an optimized assay with a substrate previous studies we found that with subsaturating concentra- concentration of 80 AtM (i.e., conditions to measure an initial tions ofPAF the activity in LDL seemed to be the relevant one; rate). However, when we examined circumstances that are e.g., depletion of LDL slowed degradation of PAF, while likely to occur in vivo, we found a difference in their behavior removal of HDL accelerated the degradation slightly. We have (6). The t1/2 of PAF was measured in plasma at a concentra- pursued this observation by using plasma from humans with tion ofPAF of either 1 nm or 0.1 AuM and then compared with lipoprotein mutations. In abetalipoproteinemia, all of the PAF the t112 in plasma that had been depleted of LDL, HDL, or acetylhydrolase activity was in HDL, whereas in Tangier both. In all cases the residual activity, as judged by the disease all of the activity was in LDL. In both conditions the optimized assay, was more than enough to ensure that it total activity measured in an optimized assay was normal or would not be the limiting component. The result was that increased. However, when we measured the t1/2 of PAF in removal of LDL significantly prolonged the t1/2, whereas plasma, we found that it was prolonged in subjects with removal ofHDL shortened it. Thus, it appeared that the PAF abetalipoproteinemia compared to normal controls. Con- acetylhydrolase activity in LDL is the physiologically rele- versely, the t1/2 in Tangier plasma was shortened. We next vant one. These findings may be important in human pathol- demonstrated that the PAF acetylhydrolase in HDL was ogy since PAF has been found in blood during allergic recognized by an antibody to the enzyme purified from LDL, reactions (11) and in renal and hepatic disease (12-14). In the establishing that the enzyme in the two particles is the same present study, we have examined these issues further by protein. Finally, we inactivated the PAF acetylhydrolase in using plasma from subjects with mutations in the relevant isolated lipoprotein particles and then reconstituted them with lipoproteins. enzyme from the opposite particle. The reconstituted particles were used to measure the t1/2 of PAF, and we again found that the LDL particle was more efficient. We conclude that the MATERIALS AND METHODS lipoprotein environment of the PAF acetylhydrolase markedly Materials. [acetyl-3H]PAF and [alkyl-3H]PAF were pur- influences its catalytic behavior. This may be important in chased from New England Nuclear. Polyclonal goat antibod- pathophysiology and will complicate attempts to assess the role ies to human apolipoprotein B (apoB) were obtained from of this enzyme in such circumstances. Technicon. Other reagents were from Sigma. The plasma samples from normal subjects were obtained from healthy Platelet-activating factor (PAF) is a phospholipid (1-0-alkyl- volunteers. The samples from three patients with abetali- 2-acetyl-sn-glycero-3-phosphocholine) that activates plate- poproteinemia (15) were provided by Roger Illingworth lets and leukocytes, causes increased vascular permeability, (University of Oregon). Another sample from a patient with and has other effects, all at concentrations as low as 0.1 nM abetalipoproteinemia was supplied by David Wilson (Boston (reviewed in refs. 1 and 2). Mammalian plasma contains an Children's Hospital). This subject, who has not been de- enzyme, PAF acetylhydrolase (1-alkyl-2-acetylglycerophos- scribed in the literature, has typical clinical and laboratory phocholine esterase, 1-alkyl-2-acetyl-sn-glycero-3-phospho- findings. The subject with Tangier disease is a patient ofDana choline acetohydrolase, EC 3.1.1.47), that specifically cata- Wilson (University of Utah), and has typical clinical and lyzes the hydrolysis of the acetyl residue and thereby laboratory manifestations (16). abolishes the bioactivity (3-5). The PAF acetylhydrolase in Assays. PAF acetylhydrolase activity was determined as human plasma was shown by Farr et al. (3) to be associated described (1 unit = 1 Amol/hr) (9). The method for deter- with low density lipoprotein (LDL). We and others have mining the t1/2 of PAF has been described in detail (6). confirmed this finding (6-8), and we have purified the Extractions oflipids used the method of Bligh and Dyer (17), enzyme from LDL to near homogeneity (9). At physiological and thin-layer chromatography separations were performed pH, about 70% of the PAF acetylhydrolase activity is by using solvent system II of Mueller et al. (18). associated with LDL, and the remainder is associated with Analyses of the Lipoprotein Location(s) of the PAF Acetyl- high density lipoprotein (HDL) (6). The activity can exchange hydrolase Activity. The isolation of HDL- and LDL-

The publication costs of this article were defrayed in part by page charge Abbreviations: PAF, platelet-activating factor; LDL., low density payment. This article must therefore be hereby marked "advertisement" lipoprotein; HDL, high density lipoprotein; DFP, diisopropyl fluo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. rophosphate; apoB, apolipoprotein B. 2393 Downloaded by guest on September 28, 2021 2394 Medical Sciences: Stafforini et al. Proc. Natl. Acad. Sci. USA 86 (1989)

associated PAF acetyihydrolase activities was carried out by excluded the possibility that either HDL or LDL is required ultracentrifugation in KBr gradients as described (6). Frac- for the activity of the acetylhydrolase to be expressed. This tions (1.5 ml) were collected and assayed. Transfer of PAF result also confirms our previous conclusion (6) that the PAF acetylhydrolase from HDL to LDL and from LDL to HDL acetylhydrolase is not a fragment of apoB-100. was carried out by dialysis at alkaline and acidic pH, The distribution of the PAF acetylhydrolase in normal respectively (6). The association of PAF acetylhydrolase plasma is 60-70% in LDL with the remainder in HDL (6). We with apoB was examined by precipitation with anti-apoB. We asked whether the distribution in the lipoprotein disorders incubated 1 1L of plasma from normal or deficient subjects was proportional to the content of the two particles since the with 10 ;LI of goat anti-apoB, in a total volume of 20 /A, for activity can exchange between them (6). We found that the 2 hr at 370C. The antiserum had been previously treated with PAF acetylhydrolase activity was localized only in LDL diisopropyl fluorophosphate (DFP; 10 mM, 60 min at 370C) to and/or HDL (Fig. 1). In the subjects with abetalipoprotein- inactivate the PAF acetylhydrolase present in goat serum. emia (who lack LDL), all of the activity was located at a Excess DFP was removed by overnight dialysis against 50 density almost identical to that ofnormal HDL (Fig. 1A). The mM Tris-HCI (pH 7.5). After the incubation with the anti- minor difference in the densities (Fig. 1, A versus B) was apoB, 30 of protein A-Sepharose 6MB [50:50 suspension observed in each of the four subjects and was reproducible. in 50 mM Tris HCl (pH 7.5)] were added, and the incubations The difference was likely due to the altered composition of were continued for an additional 2 hr at 370C, with rocking. HDL that has been described in abetalipoproteinemia (10). The amount ofPAF acetylhydrolase activity was determined To establish whether this particle was the same as the HDL in the supernatants after removal of the protein A-Sepharose that contains PAF acetylhydrolase in normal plasma, we beads by centrifugation. Control experiments demonstrated performed two experiments. In the first, we examined that control DFP-treated goat serum and DFP-treated anti- whether this particle, like those in normal plasma, could apoB had no effect by themselves on acetylhydrolase activ- donate its acetylhydrolase activity to LDL (6). Plasma from ity. In addition, the enzyme did not interact directly with subjects with abetalipoproteinemia was incubated at pH 9.0 protein A-Sepharose. We have previously shown that the with normal plasma (that had been pretreated with DFP to precipitation of PAF acetylhydrolase activity by this anti- inactivate the endogenous acetylhydrolase). Just as with body is based on the recognition ofthe apolipoprotein and not normal HDL serving as a donor particle, the particle in the enzyme was car- the (6). Heparin-agarose chromatography abetalipoproteinemia plasma served as a donor to LDL (data ried out as previously described (6), except that a step elution not shown). The second piece of evidence that the high was used instead of a gradient. density particle was functionally the same as that in normal Antibody to PAF Acetyihydrolase. We raised polyclonal antibodies in New Zealand White rabbits against the PAF plasma was that it bound to heparin-agarose and then was eluted at the same concentration of NaCl (6) as activity from acetylhydrolase purified from human LDL, following a a normal This is since multiple intradermal injection protocol (19). Rabbit sera were sample. particularly discriminating assessed for the presence of anti-PAF acetylhydrolase by only HDL that contains apoE has this trait (10), and we have Western blot analysis and by the ability of the antisera to shown previously that it is this small subset of HDL that precipitate activity. The antiserum used in the studies re- contains all of the PAF acetylhydrolase (6). ported here will be described in more detail elsewhere. The converse lipoprotein abnormality, the virtual absence However, it precipitates the pure PAF acetylhydrolase and of HDL, demonstrated the opposite localization pattern to that still incorporated into an LDL particle, but it does not that in LDL deficiency. In the plasma of a subject with precipitate apoB-100 alone. Tangier disease, we found that all ofthe PAF acetylhydrolase activity localized with LDL (Fig. 1C). To demonstrate that this particle was LDL, we performed the converse of the RESULTS transfer experiment described above and found that it func- Characterization of PAF Acetylhydrolase Activity in Sub- tioned normally as a donor. That is, the activity transferred jects with Abnormal Lipoproteins. We assayed the activity of from the Tangier LDL-like particle to normal HDL at pH = PAF acetylhydrolase in the plasma of normal subjects and 6.0 (data not shown). We also incubated the Tangier plasma subjects with abetalipoproteinemia (Table 1). The total ac- with a polyclonal antibody to apoB-100 and showed that the tivity, as measured in the optimized assay with a saturating acetylhydrolase activity was coprecipitated with apoB-100 concentration of substrate, was the same. Plasma from a (Fig. 2). The anti-apoB-100 employed did not react directly subject with Tangier disease had an activity of 4.5 units/ml. with the acetylhydrolase since it did not precipitate any The findings in the plasmas with lipoprotein deficiencies activity in the sample from the subject with abetalipopro- Table 1. Total PAF acetyihydrolase activity and the t1/2 of PAF in the plasma of subjects with lipoprotein deficiencies PAF acetylhydrolase, units/ml t1/2, min Subjects n Mean ± SD Range Mean ± SD Range Normal 6 2.5 ± 0.4 2.1-3.1 7.8 ± 1.6 6.1-10.2 Abetalipoproteinemia 4 2.6 ± 0.9 1.8-3.9 11.6 ± 0.8* 10.5-12.4 The average value shown for the normal subjects, whose plasma was assayed simultaneously with that from subjects with abetalipoproteinemia, is the same as we have determined in a population study in which over 400 individuals of either gender and from the second through the seventh decade have been tested. The value for each subject is the mean of two experiments, each performed in triplicate. The variation in each individual was less than 5%. The t1/2 of PAF in plasma was determined as described in Materials and Methods and ref. 6. Each set of assays contained a normal control as an internal standard. In four separate experiments the t1/2 of PAF in the plasma of the normal subject shown in Fig. 3 (and included in the normal sample here) was 8.5 ± 0.9 min, indicating that the assay was reproducible. *Different from the control (P < 0.01). Downloaded by guest on September 28, 2021 Medical Sciences: Stafforini et A Proc. Nati. Acad. Sci. USA 86 (1989) 2395

5000 was rate-limiting, the enzyme in LDL appeared to be more A Abetalipoproteinemia active than that in HDL (6). The naturally occurring human 4000 mutations offered an opportunity to examine this issue further. We measured the tl/2 ofsubsaturating concentrations 3000 ofPAF in whole plasma. This assay reflects different aspects * 1. of the behavior of the acetyihydrolase than the optimized 1 2000 i I. assay. In the tl/2 assays there is a substrate concentration (10 K. nM) well below saturation, an excess of albumin and other 1000 lipid-binding materials, and an excess of enzyme. These a. conditions are likely to represent the situation in vivo, where E 0 only low concentrations of PAF have been detected and the C.) 4000 other conditions also pertain. That the vastly different con- B Normal ditions under which the two assays are performed do affect 0 acetylhydrolase activity was shown by comparing the rate of - co 3000 hydrolysis in the t1/2 assay with that predicted from the kinetic constants determined in the optimized assay with 1... zco0 2000- normal plasma as a source ofenzyme (Km of5.7 AuM and Vma 1... of 1.3 itmol min-1nml-1; ref. 9). At the concentration of PAF (10 nM) used in the t1/2 assay, the predicted rate from the 0 1000 - kinetic constants was 9.5 pmol/min, while the most rapid rate 0 4"HDL LDL measured in the t1/2 experiments was 0.16 pmol/min. Thus, U.. in normal plasma, measurement of the rates of PAF hydrol- '00 ysis with the two different assays yielded rates that differed a. C N by 14 Tangier 60-fold. 40100 The t1/2 of PAF in plasma was prolonged, as compared to 00~~~~~~~~~~~~~~~~~~~~~~~~~1 normal subjects, in each subject with abetalipoproteinemia, 30 where the acetylhydrolase activity is exclusively associated with HDL (Fig. 3). The correlation of functional activity in 00700~-~ ~ ~~~~ 201 this assay and the location of the enzyme was further - 00~ ~ ~A 4- ~~~ strengthened by the finding of a shorter than normal t1/2 in 101 Tangier disease, where all of the acetylhydrolase activity is associated with LDL (Fig. 3). To exclude the possibility ofan

0 I inhibitor in the plasma ofsubjects with abetalipoproteinemia, 0 1 0 20 3 0 we determined the tl/2 of PAF in a mixture of equal amounts Fraction Number of plasma from a normal and abetalipoproteinemic subject. The mixture gave an intermediate value (normal = 7.8 min; FIG. 1. Distribution of PAF acetylhydrolase activity in normal mixture = 9.0; mutant = 10.2), indicating that there was no plasma (B) and in plasma deficient in LDL (A) or HDL (C). Plasma inhibitor present. was obtained from subjects with normal lipoproteins, abetalipopro- These results strongly supported our previous conclusion teinemia, or Tangier disease. The plasma samples were fractionated that the in by centrifugation in a density gradient formed with KBr, and the PAF environment which the PAF acetylhydrolase exists acetylhydrolase activity was assayed (6). The results are expressed influences its catalytic activity (6). This conclusion is valid as the radioactivity released as acetate in each assay. Each ofthe four only if the activities in LDL and HDL are due to the same subjects with abetalipoproteinemia had a profile like the one shown. enzyme. We have presented a comparison of the activity in intact HDL with the purified enzyme from LDL that strongly teinemia (Fig. 2). Thus, the PAF acetylhydrolase in a Tangier 100 patient was associated with LDL. The PAF Acetylhydrolase Activity Is Influenced by Its Environment. Our previous studies with the PAF acetylhydrolase had shown that when the concentration of substrate C 5 ErUX Abet 3 No antibody E EJ Control antibody = Anti-apoB antibody UA. Normal

100

4) 80 Tangier 2' 60 10 I

0 0 5 10 15 Time, min 20 FIG. 3. t1/2 of PAF in plasma from normal subjects or subjects 00 with Tangier disease or abetalipoproteinemia. We added [alkyl- Normal Abeta Tangier 3H]PAF (10 nM) to plasma from subjects with normal lipoproteins, abetalipoproteinemia (Abeta), or Tangier disease (25°C). Aliquots FIG. 2. Precipitation of PAF acetylhydrolase activity in LDL were removed at the times shown, and lipids were extracted and with antibodies to apoB. Plasma from normal, abetalipoproteinemic separated by TLC. The PAF and 1-alkyl-sn-glycero-3-phospho- (Abeta), or Ta~ngier disease subjects was incubated with anti-apoB or choline (lyso PAF) were recovered, and the radioactivity was control antibody. The antigen-antibody complexes were removed determined by liquid scintillation spectrometry. The results shown with protein A-Sepharose, and the PAF acetylhydrolase activity are representative ofall four subjects with abetalipoproteinemia and remaining in the supernatant was determined. six normal subjects. Downloaded by guest on September 28, 2021 2396 Medical Sciences: Stafforini et al. Proc. Natl. Acad. Sci. USA 86 (1989) supports this conclusion (6, 9). To further examine this point, we tested the ability of an antiserum raised against the PAF acetyihydrolase purified from LDL (9) to precipitate the activity associated with HDL (Table 2). The antibody under 0~~~~50 R-LDL the condition employed did not directly inhibit LDL- or HDL-associated enzyme activity (data not shown). In the presence of protein A, the HDL activity, like the LDL- associated activity, was precipitated by the specific antiserum but not by control serum. From this result and the U. X previously presented data, we conclude that the activities LDL present in the two particles are due to the same enzyme. 10- We next asked whether we could reproduce the effects of 0 5 10 15 the lipoprotein environment on the t1/2 of PAF by manipu- Time, min lations in vitro that would reproduce the abnormal distribution of activity observed in the patient plasmas. We isolated FIG. 4. Effect oflipoprotein environment on the catalytic behav- LDL and HDL particles from normal subjects by density ior of PAF acetylhydrolase. HDL and LDL were isolated, and gradient centrifugation, and, in portions of these prepara- portions were treated with 10 mM DFP to inactivate the PAF tions, we inactivated the PAF we acetylhydrolase (6). The DFP-treated LDL particle was incubated acetylhydrolase. Then, with HDL at pH 9.0 for 60 min at 370C. In parallel, the DFP-treated reconstituted the inactivated particle by transfer from an HDL particle was incubated with LDL at pH 6.0. The procedure active particle of the opposite type. Thus, after the transfer carried out at pH 9 results in the repletion of LDL with PAF step, LDL contained an inactivated endogenous acetylhy- acetylhydrolase from HDL (R-LDL). Conversely, the transfer car- drolase and an active acetylhydrolase that had been trans- ried out at pH 6 results in the repletion of HDL with enzyme from ferred from HDL. The reconstituted HDL particle had the LDL (R-HDL). The individual lipoproteins were reisolated and used opposite composition. The particles were repurified by ul- as the enzyme source in t1/2 experiments ([PAF] = 0.1 ;LM). Each tracentrifugation, and the redistribution of the acetylhydro- incubation contained 0.24 unit of enzyme, as measured in the lase activity into the acceptor particles was confirmed. The optimized assay. reconstituted particles and the native LDL and HDL particles from the same donor were used as a source of PAF great excess in all circumstances. The findings in the abnor- acetylhydrolase to determine the t1/2 of PAF. Each incuba- mal plasmas are supported by the reconstitution assays with tion contained the same amount of activity, as measured in normal HDL and LDL. We also have shown here that the the optimized enzymatic assay. We found (Fig. 4) that the PAF acetylhydrolase in HDL crossreacts immunologically reconstituted enzyme behaved like that originally in the with that from LDL, which supports our previous conclusion acceptor particle, not like the donor particle from which it that they are the same protein. was derived. That is, the catalytic efficiency of the PAF The physiological or pathological significance of the influ- acetylhydrolase under these conditions was determined by ence of the lipoprotein environment on the PAF acetylhy- the lipoprotein environment. drolase is not known. LDL has been shown to block the activation of neutrophils by urate crystals (20) and to de- crease the production of a procoagulant by monocytes (21). DISCUSSION Also, plasma contains a lipoprotein-associated inhibitor of The central finding in these studies is that the plasma PAF tissue factor and coagulation factor X (22, 23). Thus, there is acetylhydrolase is more active against subsaturating concen- precedence for the association of anti-inflammatory and trations of PAF if it is in LDL rather than HDL. The t1/2 antithrombotic proteins with lipoproteins, and the PAF values of PAF in the plasma of subjects with mutations that acetylhydrolase could provide both such activities by de- result in a lack of either LDL or HDL support this conclu- grading PAF. The prolongation of the t1/2 of PAF in the sion. In abetalipoproteinemic plasma, which lacks LDL, all plasma of subjects with abetalipoproteinemia should result in of the PAF acetylhydrolase was in HDL, and the t1/2 was a higher steady-state concentration of PAF in their plasma prolonged as compared to normal values. In plasma from a under circumstances in which it is released from cells. It is subject with Tangier disease, the opposite was true-since not clear how important this might be since some cells [e.g., HDL was absent (or very low), all ofthe enzyme was in LDL, endothelial cells (24-27) and neutrophils (27, 28)] release little and the t112 was shorter. These results were independent of or none of the PAF that they synthesize. However, mono- the total amount ofenzyme activity in plasma, as determined cytes release a substantial fraction ofthe PAF they make (29), in the optimized enzymatic assay, since it was present in as do eosinophils (30), and the presence of PAF in biological fluids clearly demonstrates that some cells release it in vivo Table 2. Precipitation of the HDL-associated PAF (11-i4, 31). We currently envision PAF as commonly func- acetylhydrolase with an antibody to the LDL-associated activity tioning on the cell surface, as in endothelial cells (25, 32), or Lipoprotein perhaps intracellularly (27) and only on occasion as a fluid- fraction Addition % activity phase mediator. In this scheme the plasma PAF acetylhy- HDL None 100* drolase would be a defense against widespread activation of Anti-PAF acetylhydrolase 6 inflammatory cells in the blood by fluid-phase PAF. If this is Preimmune serum 71 the role for the PAF acetylhydrolase, then the decreased LDL None loot efficiency when localized to HDL (e.g., in abetalipoprotein- Anti-PAF acetylhydrolase 18 emia) may be manifest only when PAF accumulates in plasma Preimmune serum 91 [e.g., allergic reactions (11) or endotoxemia (33, 34)]. An- The indicated lipoprotein fractions were incubated with PAF other circumstance in which the plasma PAF acetylhydrolase acetylhydrolase antiserum or preimmune serum and then with may play an important role is in the regulation ofthe onset of protein A-Sepharose. The supernatants then were assayed for PAF labor, since Maki et al. (35) have shown a dramatic fall in the acetylhydrolase activity. level of enzymatic activity in the maternal plasma of rabbits *Activity was 0.08 unit. just at the time of labor. Whether this change is relevant to tActivity was 0.05 unit. human biology is not clear since the enzyme is entirely Downloaded by guest on September 28, 2021 Medical Sciences: Stafforini et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2397

localized to HDL in rabbits (ref. 35; unpublished observa- 8. Ostermann, G., Kertscher, H.-P., Winkler, L., Schlag, B., tions). Ruhling, K. & Till, U. (1986) Thromb. Res. 44, 303-314. The activity of other enzymes, particularly phospholi- 9. Stafforini, D. M., Prescott, S. M. & McIntyre, T. M. (1987) J. Biol. Chem. 262, 4223-4230. pases, has been shown to be affected by the lipid environment 10. Mahley, R. W., Innerarity, T. L., Rall, S. C. & Weisgraber, (36, 37). In the case ofphospholipases, the physical nature of K. H. (1984) J. Lipid Res. 25, 1277-1294. the phospholipids influences activity, as does the molecular 11. Grandel, K. E., Farr, R. S., Wanderer, A. A., Eisenstadt, species (37). The current description is the clearest example T. C. & Wasserman, S. I. (1985) N. Engl. J. Med. 313, 405- of the modulation of the activity of a phospholipase by its 409. native environment. The molecular mechanism by which 12. Caramelo, C., Fernandez-Gallardo, S., Marin-Cao, D., Ifiar- rea, P., Santos, J. C., Lopez-Novoa, J. M. & Sanchez-Crespo, PAF acetyihydrolase activity is modulated in HDL versus M. (1984) Biochem. Biophys. Res. Commun. 120, 789-7%. LDL is not clear. It is conceivable that the apolipoprotein, 13. Ito, S., Camussi, G., Tetta, C., Milgrom, F. & Andres, G. the lipids, or both could be responsible. (1984) Lab. Invest. 51, 148-161. Pritchard and coworkers (7) found that a patient with 14. Caramelo, C., Fernandez-Gallardo, S., Santos, J. C., Ifiarrea, Tangier disease had increased PAF acetylhydrolase activity P., Sanchez-Crespo, M., Lopez-Novoa, J. M. & Hernando, L. similar to what we found. Ostermann et al. (8) used two (1987) Eur. J. Clin. Invest. 17, 7-11. different assays to examine the ability ofisolated lipoproteins 15. Illingworth, D. R., Alam, S. S. & Alam, N. A. (1983) Metab- to catalyze the hydrolysis ofPAF. They measured the release olism 32, 869-873. of radiolabeled acetate from PAF, an assay similar to our 16. Herbert, P. N., Assmann, G., Gotto, A. M. & Fredrickson, D. S. (1983) in The Metabolic Basis ofInherited Disease, eds. optimized assay except that their concentration of substrate Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S., Gold- (11 uM) was just at the Km (9) instead of severalfold higher. stein, J. L. & Brown, M. S. (McGraw-Hill, New York), 5th They also measured loss of bioactivity when PAF was Ed., pp. 589-621. incubated for 30 min with plasma or isolated lipoproteins and 17. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, found that HDL was less effective than LDL (8). 911-917. One implication ofour findings is that studies of the role of 18. Mueller, H. W., O'Flaherty, J. T. & Wykle, R. L. (1983) J. the PAF acetylhydrolase in diseases should consider both the Biol. Chem. 258, 6213-6218. activity as measured as an initial rate in an enzyme assay and 19. Vaitukaitis, J. L. (1981) Methods Enzymol. 73, 46-52. the of PAF in plasma. For example, it has been reported 20. Terkeltaub, R., Curtiss, L. K., Tenner, A. J. & Ginsberg, t112 M. H. (1984) J. Clin. Invest. 73, 1719-1730. that increased total activity is associated with hypertension, 21. Schwartz, B. S., Levy, G. A., Curtiss L. K., Fair, D. S. & presumably as a result of the removal of some constitutively Edgington, T. S. (1981) J. Clin. Invest. 67, 1650-1658. synthesized PAF that was exerting an antihypertensive effect 22. Broze, G. J. & Miletich, J. P. (1987) Blood 69, 150-155. (38). However, our finding suggests that the higher acetyl- 23. Broze, G. J., Warren, L. A., Novotny, W. F., Higuchi, D. A., hydrolase activity measured under optimal conditions might Girard, J. A. & Miletich, J. P. (1988) Blood 71, 335-343. not necessarily result in a lower concentration of PAF in 24. McIntyre, T. M., Zimmerman, G. A., Satoh, K. & Prescott, plasma. In summary, we have demonstrated that the activity S. M. (1985) J. Clin. Invest. 76, 271-280. of the plasma enzyme that specifically degrades PAF is 25. McIntyre, T. M., Zimmerman, G. A. & Prescott, S. M. (1986) influenced the lipoprotein environment in which Proc. Natl. Acad. Sci. USA 83, 2204-2208. strongly by 26. Lewis, M. L., Whatley, R. E., Cain, P., McIntyre, T. M., it is located. The fraction that is in LDL, about 70o of the Prescott, S. M. & Zimmerman, G. A. (1988) J. Clin. Invest. 82, total in normal subjects, is the most active under conditions 2045-2055. that are likely to be relevant in vivo. 27. Lynch, J. M. & Henson, P. M. (1986) J. Immunol. 137, 2653- 2661. We are grateful to Drs. Roger Illingworth, David Wilson, and Dana 28. Sisson, J. H., Prescott, S. M., McIntyre, T. M. & Zimmerman, Wilson for generously providing us samples of plasma from their G. A. (1987) J. Immunol. 138, 3918-3926. patients. Bart Tarbet contributed excellent technical assistance. We 29. Elstad, M. R., Prescott, S. M., McIntyre, T. M. & Zimmer- thank Dr. Lily Wu for analyzing the lipoproteins in the samples. man, G. A. (1988) J. Immunol. 140, 1618-1624. Linda Jara and Leona Archuleta helped prepare the manuscript. This 30. Lee, T.-C., Lenihan, D. J., Malone, B., Roddy, L. L. & work was supported by the Nora Eccles Treadwell Foundation and Wasserman, S. I. (1984) J. Biol. Chem. 259, 5526-5530. by a grant from the National Institutes ofHealth (HL 35828). S.M.P. 31. Billah, M. M. & Johnston, J. M. (1983) Biochem. Biophys. Res. and G.A.Z. are Established Investigators of the American Heart Commun. 113, 51-58. Association. 32. Zimmerman, G. A., McIntyre, T. M. & Prescott, S. M. (1985) J. Clin. Invest. 76, 2235-2246. 1. Snyder, F. (1985) Med. Res. Rev. 5, 107-140. 33. Doebber, T. M., Wu, M. S., Robbins, J. C., Choy, B. M., 2. Hanahan, D. J. (1986) Annu. Rev. Biochem. 55, 483-509. Chang, M. N. & Shen, T. Y. (1985) Biochem. Biophys. Res. 3. Farr, R. S., Wardlow, M. L., Cox, C. P., Meng, K. E. & Commun. 127, 799-808. Greene, D. E. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 34. Chang, S.-W., Fedderson, C. O., Henson, P. M. & Voelkel, 3120-3122. N. E. (1987) J. Clin. Invest. 79, 1498-1509. 4. Wardlow, M. L., Cox, C. P., Meng, K. E., Greene, D. E. & 35. Maki, N., Hoffman, D. R. & Johnston, J. M. (1988) Proc. Natl. Farr, R. S. (1986) J. Immunol. 136, 3441-3446. Acad. Sci. USA 85, 728-732. 5. Blank, M. L., Lee, T.-C., Fitzgerald, V. & Snyder, F. (1981) J. 36. Spector, A. A. & Yorek, M. A. (1985) J. Lipid Res. 26, 1015- Biol. Chem. 256, 175-178. 1035. 6. Stafforini, D. M., McIntyre, T. M., Carter, M. E. & Prescott, 37. Dennis, E. A. (1983) in The Enzymes: Lipid Enzymology, ed. S. M. (1987) J. Biol. Chem. 262, 4215-4222. Boyer, P. D. (Academic, New York), 3rd Ed., pp. 308-353. 7. Pritchard, P. H., Chonn, A. & Yeung, C. C. (1985) Blood 66, 38. Blank, M. L., Hall, M. N., Cress, E. A. & Snyder, F. (1983) 1476-1478. Biochem. Biophys. Res. Commun. 113, 666-671. Downloaded by guest on September 28, 2021