[14]
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[ 14] B i o a s s a y o f p a f - A c e t h e r b y R a b b i t P l a t e l e t A g g r e g a t i o n
By MARIE-JEANNE BOSSANT, EWA N I N I O ,
DANII~LE DELAUT1ER, JACQUES BENVENISTE
and
Three basic methods exist for accurate quantitation of chemical mediators: (1) measurement of their biological activity, (2) immunological identity, and (3) physicochemical properties, such as chromatographic mobility and mass spectrometric behavior. In the case of paf-acether (paD, our method of choice is a bioassay ~-9 since the immunoassays that are now being developed have not yet proved their usefulness in day-to-day use in the research laboratory. Physicochemical methods are hampered by the very low paf concentration typically encountered. The present biological method for quantitation of paf is based on the monitoring of platelet activation either assessing platelet aggregation in an aggregometer or monitoring the release of radiolabeled serotonin previously incorporated into platelets. Several criteria are used to distinguish paf from other platelet agonists: (1) conditions of platelet activation, (2) normal and reversedphase chromatographic elution behavior identical to that of synthetic paf, (3) inactivation by phospholipases: A2, C, and D, and resistance to lipase from Rhizopus arrhizus, and (4) when enough of the mediator can be recovered, gas chromatography and mass spectrometry characteristics. Other methods, less frequently used, have been described, e.g., the platelet-desensitizing activity of paf or, for some cellular species, the incorporation of [3H]acetate into paf. J. Benveniste, P. M. Henson, and C. Cochrane, J. Exp. Med. 316, 1356 (1972). 2 j. Benveniste, Nature (London) 249, 581 (1974). 3 j. Benveniste, J. p. Le Couedic, J. Polonsky, and M. Tenc6, Nature (London) 269, 170 (1977). 4 p. M. Henson, J. Clin. lnvest. 60, 481 (1977). 5 j. p. Cazenave, J. Benveniste, and J. F. Mustard, Lab. Invest. 41, 275 (1979). 6 C. A. Demopoulos, R. N. Pinckard, and D. J. Hanahan, J. Biol. Chem. 254, 9355 (1979). 7 M. L. Blank, F. Snyder, L. W. Byers, B. Brooks, and E. E. Muirhead, Biochem. Biophys. Res. Commun. 911, 1191 (1979). 8 j. Benveniste, M. Tenc6, P. Varenne, J. Bidault, C. Boullet, and J. Poionsky, C.R. Acad. Sci. (Paris) 289D, 1037 (1979). 9 j. Polonsky, M. Tenc6, P. Varenne, B. C. Das, J. Lunel, and J. Benveniste, Proc. Natl. Acad. Sci. U.S.A. 77, 7019 (1980).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
126
ASSAYS
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Platelet Aggregation Method 1,5 Preparation o f Washed Rabbit Platelets This method, a modification of that of Ardlie et al., lo is based on the differential centrifugation of whole blood since platelets have the lowest density and thus can be readily separated from leukocytes and erythrocytes. Reagents Ethylenediaminetetraacetate (EDTA), 0.2 M, pH 7.2 Tyrode's gelatin buffer devoid of Ca 2÷ (TG no Ca2÷-EGTA), pH 6.5, in mM: 2.6 KCI, 1.05 MgCI~, 137 NaCI, 12.1 NaHCO3, 5.6 glucose, 0.2 EGTA { [ethylenebis(oxyethylenenitrilo)]tetraacetic acid} and 0.25% gelatin (w/v) Acetylsalicylic acid (Aspegic, Egic Lab, Amilly, France), 100 mM Procedure. Adult New Zealand White rabbits are bled from the ear artery (40 ml) into a Falcon tube containing 1 ml of 0.2 M EDTA. Platelets are obtained using a modification of the method of Ardlie et al. 10Blood is centrifuged at room temperature at 400 g for 20 min to allow separation into three layers: (1) the platelet-rich plasma, (2) the buffy coat, and (3) the erythrocyte and neutrophil layers. The platelet-rich plasma is recovered, treated with acetylsalicylic acid (0. I mM, final concentration) for 30 min at room temperature, 5 and platelets are separated from plasma by centrifugation at 2000 g for 20 min. The platelet pellet is washed twice following resuspension in TG no Ca2+-EGTA (pH 6.5) and centrifugation. Platelets are finally suspended in this same medium and kept at room temperature. Aggregation o f Platelets with p a f Platelet aggregation is monitored using an aggregometer, the principle of which is based on the measurement of light transmission through the platelet suspension (this physical phenomenon is termed turbidimetry). Aggregated platelets will allow proportionally higher light transmission than nonaggregated ones. Reagents Tyrode's gelatin buffer with calcium (TG), in mM: 2.6 KCI, 1 MgCI2, 137 NaCI, 12.1 NaHCO3, 5.6 glucose, 0.9 CaCI2 and 4.2 HEPES (N-2-
1o N. G. Ardli¢, M. A. Packham, and J. F. Mustard, Br. J. H a e m a t o l . 19, 7 (1970).
[14]
BIOASSAY OF paf-ACETHER: PLATELET AGGREGATION
127
hydroxyethylpiperazine-N'-2-ethanesulfonic acid, Calbiochem, San Diego, CA), 0.25% gelatin (w/v), pH 7.4. H E P E S - B S A buffer, in mM: 4.2 HEPES, 137 NaCI, 2.6 KCI, 0.65 CaCI2, 0.5 MgCI2, and 0.25% fatty acid-free bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) C16:0 PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) or C18:0 PAF (1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) (Novabiochem, Clery en Vexin, France) Creatine phosphate 133 mM/creatine phosphokinase 1333 U/ml (CP/ CPK) (Sigma), stock solutions in 0.15 M NaCI are kept in aliquots (300/xl) at - 2 0 ° Procedure
PREPARATION OF STANDARD p a f SOLUTIONS. Weigh 4.408 mg C18:0 paf or 4.192 mg C16:0 paf into a glass tube. Dissolve in 1 ml CH2Cl2 : CH3OH (l : 1), paf concentration: 8 mM (sample I). Take 50/xl and evaporate under air stream. Add 2 ml CH2CI2 :CHzOH, 1 : 1 (v/v), pal concentration: 0.2 mM (sample 2). (It was empirically found that evaporation and resuspending paf in CH2C12 :CHaOH, gives better yield than simply dissolving in the same solution.) Take 50 /xl of solution 2 and evaporate. Add 2 ml CH2C12 :CH3OH 1:1, paf concentration: 5 /xM (sample 3). Take 200/xl of solution 3 and evaporate. Add I ml NaC! 0.15 M with 0.25% BSA (w/v), paf concentration: 1 nM (sample 4). From sample 4, four other concentrations of standard paf are prepared by dilution with NaCI-BSA: 0.8, 0.6, 0.4, and 0.2 nM. SAMPLE PREPARATION. Samples containing paf are prepared as follows. Cells with their medium (to assess the total formation of paf) or cells and medium separately (to assess cell-associated vs. released pat') are extracted with 80% ethanol (final concentration) and continuously mixed for 1 hr at 20°. The extracts are centrifuged, the pellets are discarded, and the supernatants brought to dryness under an air stream at 40°. Dry residues containing paf are then suspended in HEPES-BSA. Tissues or E s c h e r i c h i a coli 11-14 are extracted according to a modified Bligh and Dyer technique) 5 Briefly, 1 volume CH2C12 and 2 volumes
tl F. Snyder,Med. Res. Reo. S, 107 (1985). J2 E. Ninio, in "New Horizonsin PlateletActivatingFactor Research" (L. M. Winslowand M. L. Lee, eds.), p. 27. Wiley,New York, 1987. f3 j. Benveniste,in "BiologicalMembranes:Aberrationsin MembraneStructureand Function" (M. L. Karnovsky,A. Leaf, and L. C. Bolis, eds.), p. 73. Liss, New York, 1988. 14y. Denizot, E. Dassa, H.-Y. Kim, M. J. Bossant, N. Salem Jr., Y. Thomas, and J. Benveniste,FEBS Lett. 243, 13 (1989). 15E. G. Blighand W. J. Dyer, Can. J. Biochem. Physiol. 37,911 (1959).
128
ASSAYS
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CH3OH are added to 1 volume aqueous cells solutions acidified at pH 3. This mixture is incubated for 24 hr at 4 ° with shaking. CH2C12 and H20 1 : 1 (v/v) are added to achieve phase separation and the lower phase is collected and evaporated to dryness under nitrogen. Lyso-paf is quantified after chemical acetylation into paf. J6 Briefly, the dry residues of the ethanolic samples are treated overnight at room temperature with 100/zl acetic anhydride and 100/zl pyridine. After evaporation of the reagents, the dry residue is washed three times with I ml of CH2C12 and is then suspended in the H E P E S - B S A described above; the paf is then assayed. The amount of lyso-paf is measured as the difference between the amounts of paf measured after and before acetylation of the samples. The yield of this method is 80%. 16 The cell content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine is measured as paf after alkaline hydrolysis, followed by acetylation. The ethanolic samples are brought to dryness and are treated with 0.03 N NaOH in methanol for 2 hr at 22 °. The pH is then adjusted to 7.4 with 1 N HC1 and the mixture brought to dryness; the dry residue is then acetylated as described above, and then bioassayed for paf activity. The yield of alkaline hydrolysis is 99.1%. 16 CALIBRATION OF PLATELETAGGREGATION.Platelets (2 × 109/ml) are stirred in 350/~l TG. To avoid the possibility that either arachidonic acid metabolites or ADP might be responsible for platelet aggregation, platelets are preincubated with acetylsalicylic acid during purification (see above) and the complex CP/CPK (300/.d of each, final concentration: 1 mM and 10 U/ml, respectively) is added to 40 ml aggregation buffer. 5 A calibration curve is obtained daily using 5/~1 of the five dilutions of C16:0 or C18:0 p a l Linear regression is applied to the experimental data (Fig. I) and the following equations are typically obtained: y = 0.92 x - 3.34, R = 0.98 and y = 0.43 x - 2.41, R = 0.95 for C16 : 0 and C 18:0 paf, respectively. Sensitivity of platelets may vary from batch to batch and require a different range of paf dilutions to establish the calibration curve. TESTING FOR PRESENCE OF PLATELET AGGREGATION INHIBITOR. Five microliters of the sample and 5/.d of pal standard solution (a concentration taken from the linear regression calibration curve that induces about 75-80% aggregation) are added to the platelet solution. If standard pal aggregation is not diminished, the sample can be quantified directly without purification. If it is decreased, purification (including thin-layer chromatography and/or liquid chromatography) is necessary. 16 E. Jouvin-Marclae, E. Ninio, G. Beaurain, M. Tenc6, P. Niaudet, and J. Benveniste, J. lmmunol. 133, 892 (1984).
[14l
BIOASSAY OF paf-ACETHER" PLATELET AGGREGATION
129
/( °
.~ .~_
.7'
40
J
E ~
.-~
T 20
~
.1
0
0
25
.
.
,
i
i
i
i
50
75
1O0
125
PAF-acether (pM) FIG, i. Calibration plot for paf bioassay. Washed rabbit platelets were challenged with paf C16:0 (0) or paf C18:0 (O). Results in percentage of light transmission are means +- SEM of three to four determinations using independent platelet preparations.
CRITERIA FOR pal IDENTIFICATION. |7 To increase the specificity of this biological method, the aggregation is verified under different conditions: paf inactivation by phospholipases A2, C, and D 3 o r inhibition of its effects by paf antagonists such as BN 52021, kadsurenone, or CV 3988. By contrast, lipase A from R. arrhizus is devoid of any inactivation effect. It is also possible to analyze the molecular species of pal using reversed-phase liquid chromatography. Collected fractions corresponding to C16:0 or C18 : 0 or other paf analogs can be thus quantified using the bioassay. ~8
Conclusions The platelet aggregation method is a very sensitive tool that can be used to detect as little as picogram quantities of paf. When using five different platelet preparations the variation coefficient was 23%. The bioassay specificity using crude biological extracts is obviously not very good but it reaches full specificity following prior purification of the samples and using stringent criteria for paf identification. The aggregation method has various advantages over the other method based on platelet activation: the 17 j. Benveniste, J. Camussi, and J. Polonsky, Monogr. Allergy 12, 138 (1977) ~a L. Michel, Y. Denizot, Y. Thomas, F. Jean-Louis, C. Pitton, J. Benveniste, and L. Dubertret, J. Immunol. 141, 948 (1988).
130
ASSAYS
[15]
release of labeled serotonin. This latter technique uses expensive radioactive compounds which must be appropriately disposed of, necessitates a preincorporation step of the label, and, most importantly, does not yield immediate data. Off-range concentrations of paf cannot be adjusted in real time as is possible with aggregation techniques where results are immediately available, thus delaying the acquisition of valid results by several days. The aggregation method was and still is the basis for most of the studies conducted so far on this mediator, notably on the structure, the origin, the metabolism, and the physiological properties of paf. In addition, most of the paf antagonists have been developed using this test either as a screening procedure or for more complex pharmacological studies.
[15] B i o a s s a y o f P l a t e l e t - A c t i v a t i n g F a c t o r b y R e l e a s e of [3H]Serotonin
By PETER M. HENSON Introduction Platelet-activating factor (PAF) is a highly potent cell communication mediator thought to be involved in a wide variety of pathophysiological processes. Three major approaches have been taken in assaying for the molecule: bioassays, including platelet aggregation, platelet secretion, and neutrophil activation; synthetic approaches, including incorporation of [3HI acetate into the molecule; and physicochemical procedures, including mass spectrometry. Each has advantages and disadvantages. The bioassays are relatively simple, can be applied to a large number of samples, often require little workup, are highly sensitive, and by the use of appropriate standards, give reasonably quantitative results. They can be carried out with minimal equipment and are inexpensive. Their disadvantage, in general, is the potential lack of specificity (necessitating appropriate controls and/or purification), since many agents can activate platelets or neutrophils, the lack of quantitative precision that would be found in physical approaches, and the inability to obtain structural data from the assay. Rabbit platelets are particularly sensitive to stimulation by PAF and are often used as indicator cells. Thus the assay is described for these cells, although it could be adapted to ceils from humans. Platelet aggregation is more sensitive to low concentrations of the mediator than is secretion and METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOASSAY OF p a f - A C E T H E R " P L A T E L E T A G G R E G A T I O N
125
[ 14] B i o a s s a y o f p a f - A c e t h e r b y R a b b i t P l a t e l e t A g g r e g a t i o n
By MARIE-JEANNE BOSSANT, EWA N I N I O ,
DANII~LE DELAUT1ER, JACQUES BENVENISTE
and
Three basic methods exist for accurate quantitation of chemical mediators: (1) measurement of their biological activity, (2) immunological identity, and (3) physicochemical properties, such as chromatographic mobility and mass spectrometric behavior. In the case of paf-acether (paD, our method of choice is a bioassay ~-9 since the immunoassays that are now being developed have not yet proved their usefulness in day-to-day use in the research laboratory. Physicochemical methods are hampered by the very low paf concentration typically encountered. The present biological method for quantitation of paf is based on the monitoring of platelet activation either assessing platelet aggregation in an aggregometer or monitoring the release of radiolabeled serotonin previously incorporated into platelets. Several criteria are used to distinguish paf from other platelet agonists: (1) conditions of platelet activation, (2) normal and reversedphase chromatographic elution behavior identical to that of synthetic paf, (3) inactivation by phospholipases: A2, C, and D, and resistance to lipase from Rhizopus arrhizus, and (4) when enough of the mediator can be recovered, gas chromatography and mass spectrometry characteristics. Other methods, less frequently used, have been described, e.g., the platelet-desensitizing activity of paf or, for some cellular species, the incorporation of [3H]acetate into paf. J. Benveniste, P. M. Henson, and C. Cochrane, J. Exp. Med. 316, 1356 (1972). 2 j. Benveniste, Nature (London) 249, 581 (1974). 3 j. Benveniste, J. p. Le Couedic, J. Polonsky, and M. Tenc6, Nature (London) 269, 170 (1977). 4 p. M. Henson, J. Clin. lnvest. 60, 481 (1977). 5 j. p. Cazenave, J. Benveniste, and J. F. Mustard, Lab. Invest. 41, 275 (1979). 6 C. A. Demopoulos, R. N. Pinckard, and D. J. Hanahan, J. Biol. Chem. 254, 9355 (1979). 7 M. L. Blank, F. Snyder, L. W. Byers, B. Brooks, and E. E. Muirhead, Biochem. Biophys. Res. Commun. 911, 1191 (1979). 8 j. Benveniste, M. Tenc6, P. Varenne, J. Bidault, C. Boullet, and J. Poionsky, C.R. Acad. Sci. (Paris) 289D, 1037 (1979). 9 j. Polonsky, M. Tenc6, P. Varenne, B. C. Das, J. Lunel, and J. Benveniste, Proc. Natl. Acad. Sci. U.S.A. 77, 7019 (1980).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
126
ASSAYS
[14]
Platelet Aggregation Method 1,5 Preparation o f Washed Rabbit Platelets This method, a modification of that of Ardlie et al., lo is based on the differential centrifugation of whole blood since platelets have the lowest density and thus can be readily separated from leukocytes and erythrocytes. Reagents Ethylenediaminetetraacetate (EDTA), 0.2 M, pH 7.2 Tyrode's gelatin buffer devoid of Ca 2÷ (TG no Ca2÷-EGTA), pH 6.5, in mM: 2.6 KCI, 1.05 MgCI~, 137 NaCI, 12.1 NaHCO3, 5.6 glucose, 0.2 EGTA { [ethylenebis(oxyethylenenitrilo)]tetraacetic acid} and 0.25% gelatin (w/v) Acetylsalicylic acid (Aspegic, Egic Lab, Amilly, France), 100 mM Procedure. Adult New Zealand White rabbits are bled from the ear artery (40 ml) into a Falcon tube containing 1 ml of 0.2 M EDTA. Platelets are obtained using a modification of the method of Ardlie et al. 10Blood is centrifuged at room temperature at 400 g for 20 min to allow separation into three layers: (1) the platelet-rich plasma, (2) the buffy coat, and (3) the erythrocyte and neutrophil layers. The platelet-rich plasma is recovered, treated with acetylsalicylic acid (0. I mM, final concentration) for 30 min at room temperature, 5 and platelets are separated from plasma by centrifugation at 2000 g for 20 min. The platelet pellet is washed twice following resuspension in TG no Ca2+-EGTA (pH 6.5) and centrifugation. Platelets are finally suspended in this same medium and kept at room temperature. Aggregation o f Platelets with p a f Platelet aggregation is monitored using an aggregometer, the principle of which is based on the measurement of light transmission through the platelet suspension (this physical phenomenon is termed turbidimetry). Aggregated platelets will allow proportionally higher light transmission than nonaggregated ones. Reagents Tyrode's gelatin buffer with calcium (TG), in mM: 2.6 KCI, 1 MgCI2, 137 NaCI, 12.1 NaHCO3, 5.6 glucose, 0.9 CaCI2 and 4.2 HEPES (N-2-
1o N. G. Ardli¢, M. A. Packham, and J. F. Mustard, Br. J. H a e m a t o l . 19, 7 (1970).
[14]
BIOASSAY OF paf-ACETHER: PLATELET AGGREGATION
127
hydroxyethylpiperazine-N'-2-ethanesulfonic acid, Calbiochem, San Diego, CA), 0.25% gelatin (w/v), pH 7.4. H E P E S - B S A buffer, in mM: 4.2 HEPES, 137 NaCI, 2.6 KCI, 0.65 CaCI2, 0.5 MgCI2, and 0.25% fatty acid-free bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) C16:0 PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) or C18:0 PAF (1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) (Novabiochem, Clery en Vexin, France) Creatine phosphate 133 mM/creatine phosphokinase 1333 U/ml (CP/ CPK) (Sigma), stock solutions in 0.15 M NaCI are kept in aliquots (300/xl) at - 2 0 ° Procedure
PREPARATION OF STANDARD p a f SOLUTIONS. Weigh 4.408 mg C18:0 paf or 4.192 mg C16:0 paf into a glass tube. Dissolve in 1 ml CH2Cl2 : CH3OH (l : 1), paf concentration: 8 mM (sample I). Take 50/xl and evaporate under air stream. Add 2 ml CH2CI2 :CHzOH, 1 : 1 (v/v), pal concentration: 0.2 mM (sample 2). (It was empirically found that evaporation and resuspending paf in CH2C12 :CHaOH, gives better yield than simply dissolving in the same solution.) Take 50 /xl of solution 2 and evaporate. Add 2 ml CH2C12 :CH3OH 1:1, paf concentration: 5 /xM (sample 3). Take 200/xl of solution 3 and evaporate. Add I ml NaC! 0.15 M with 0.25% BSA (w/v), paf concentration: 1 nM (sample 4). From sample 4, four other concentrations of standard paf are prepared by dilution with NaCI-BSA: 0.8, 0.6, 0.4, and 0.2 nM. SAMPLE PREPARATION. Samples containing paf are prepared as follows. Cells with their medium (to assess the total formation of paf) or cells and medium separately (to assess cell-associated vs. released pat') are extracted with 80% ethanol (final concentration) and continuously mixed for 1 hr at 20°. The extracts are centrifuged, the pellets are discarded, and the supernatants brought to dryness under an air stream at 40°. Dry residues containing paf are then suspended in HEPES-BSA. Tissues or E s c h e r i c h i a coli 11-14 are extracted according to a modified Bligh and Dyer technique) 5 Briefly, 1 volume CH2C12 and 2 volumes
tl F. Snyder,Med. Res. Reo. S, 107 (1985). J2 E. Ninio, in "New Horizonsin PlateletActivatingFactor Research" (L. M. Winslowand M. L. Lee, eds.), p. 27. Wiley,New York, 1987. f3 j. Benveniste,in "BiologicalMembranes:Aberrationsin MembraneStructureand Function" (M. L. Karnovsky,A. Leaf, and L. C. Bolis, eds.), p. 73. Liss, New York, 1988. 14y. Denizot, E. Dassa, H.-Y. Kim, M. J. Bossant, N. Salem Jr., Y. Thomas, and J. Benveniste,FEBS Lett. 243, 13 (1989). 15E. G. Blighand W. J. Dyer, Can. J. Biochem. Physiol. 37,911 (1959).
128
ASSAYS
[14]
CH3OH are added to 1 volume aqueous cells solutions acidified at pH 3. This mixture is incubated for 24 hr at 4 ° with shaking. CH2C12 and H20 1 : 1 (v/v) are added to achieve phase separation and the lower phase is collected and evaporated to dryness under nitrogen. Lyso-paf is quantified after chemical acetylation into paf. J6 Briefly, the dry residues of the ethanolic samples are treated overnight at room temperature with 100/zl acetic anhydride and 100/zl pyridine. After evaporation of the reagents, the dry residue is washed three times with I ml of CH2C12 and is then suspended in the H E P E S - B S A described above; the paf is then assayed. The amount of lyso-paf is measured as the difference between the amounts of paf measured after and before acetylation of the samples. The yield of this method is 80%. 16 The cell content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine is measured as paf after alkaline hydrolysis, followed by acetylation. The ethanolic samples are brought to dryness and are treated with 0.03 N NaOH in methanol for 2 hr at 22 °. The pH is then adjusted to 7.4 with 1 N HC1 and the mixture brought to dryness; the dry residue is then acetylated as described above, and then bioassayed for paf activity. The yield of alkaline hydrolysis is 99.1%. 16 CALIBRATION OF PLATELETAGGREGATION.Platelets (2 × 109/ml) are stirred in 350/~l TG. To avoid the possibility that either arachidonic acid metabolites or ADP might be responsible for platelet aggregation, platelets are preincubated with acetylsalicylic acid during purification (see above) and the complex CP/CPK (300/.d of each, final concentration: 1 mM and 10 U/ml, respectively) is added to 40 ml aggregation buffer. 5 A calibration curve is obtained daily using 5/~1 of the five dilutions of C16:0 or C18:0 p a l Linear regression is applied to the experimental data (Fig. I) and the following equations are typically obtained: y = 0.92 x - 3.34, R = 0.98 and y = 0.43 x - 2.41, R = 0.95 for C16 : 0 and C 18:0 paf, respectively. Sensitivity of platelets may vary from batch to batch and require a different range of paf dilutions to establish the calibration curve. TESTING FOR PRESENCE OF PLATELET AGGREGATION INHIBITOR. Five microliters of the sample and 5/.d of pal standard solution (a concentration taken from the linear regression calibration curve that induces about 75-80% aggregation) are added to the platelet solution. If standard pal aggregation is not diminished, the sample can be quantified directly without purification. If it is decreased, purification (including thin-layer chromatography and/or liquid chromatography) is necessary. 16 E. Jouvin-Marclae, E. Ninio, G. Beaurain, M. Tenc6, P. Niaudet, and J. Benveniste, J. lmmunol. 133, 892 (1984).
[14l
BIOASSAY OF paf-ACETHER" PLATELET AGGREGATION
129
/( °
.~ .~_
.7'
40
J
E ~
.-~
T 20
~
.1
0
0
25
.
.
,
i
i
i
i
50
75
1O0
125
PAF-acether (pM) FIG, i. Calibration plot for paf bioassay. Washed rabbit platelets were challenged with paf C16:0 (0) or paf C18:0 (O). Results in percentage of light transmission are means +- SEM of three to four determinations using independent platelet preparations.
CRITERIA FOR pal IDENTIFICATION. |7 To increase the specificity of this biological method, the aggregation is verified under different conditions: paf inactivation by phospholipases A2, C, and D 3 o r inhibition of its effects by paf antagonists such as BN 52021, kadsurenone, or CV 3988. By contrast, lipase A from R. arrhizus is devoid of any inactivation effect. It is also possible to analyze the molecular species of pal using reversed-phase liquid chromatography. Collected fractions corresponding to C16:0 or C18 : 0 or other paf analogs can be thus quantified using the bioassay. ~8
Conclusions The platelet aggregation method is a very sensitive tool that can be used to detect as little as picogram quantities of paf. When using five different platelet preparations the variation coefficient was 23%. The bioassay specificity using crude biological extracts is obviously not very good but it reaches full specificity following prior purification of the samples and using stringent criteria for paf identification. The aggregation method has various advantages over the other method based on platelet activation: the 17 j. Benveniste, J. Camussi, and J. Polonsky, Monogr. Allergy 12, 138 (1977) ~a L. Michel, Y. Denizot, Y. Thomas, F. Jean-Louis, C. Pitton, J. Benveniste, and L. Dubertret, J. Immunol. 141, 948 (1988).
130
ASSAYS
[15]
release of labeled serotonin. This latter technique uses expensive radioactive compounds which must be appropriately disposed of, necessitates a preincorporation step of the label, and, most importantly, does not yield immediate data. Off-range concentrations of paf cannot be adjusted in real time as is possible with aggregation techniques where results are immediately available, thus delaying the acquisition of valid results by several days. The aggregation method was and still is the basis for most of the studies conducted so far on this mediator, notably on the structure, the origin, the metabolism, and the physiological properties of paf. In addition, most of the paf antagonists have been developed using this test either as a screening procedure or for more complex pharmacological studies.
[15] B i o a s s a y o f P l a t e l e t - A c t i v a t i n g F a c t o r b y R e l e a s e of [3H]Serotonin
By PETER M. HENSON Introduction Platelet-activating factor (PAF) is a highly potent cell communication mediator thought to be involved in a wide variety of pathophysiological processes. Three major approaches have been taken in assaying for the molecule: bioassays, including platelet aggregation, platelet secretion, and neutrophil activation; synthetic approaches, including incorporation of [3HI acetate into the molecule; and physicochemical procedures, including mass spectrometry. Each has advantages and disadvantages. The bioassays are relatively simple, can be applied to a large number of samples, often require little workup, are highly sensitive, and by the use of appropriate standards, give reasonably quantitative results. They can be carried out with minimal equipment and are inexpensive. Their disadvantage, in general, is the potential lack of specificity (necessitating appropriate controls and/or purification), since many agents can activate platelets or neutrophils, the lack of quantitative precision that would be found in physical approaches, and the inability to obtain structural data from the assay. Rabbit platelets are particularly sensitive to stimulation by PAF and are often used as indicator cells. Thus the assay is described for these cells, although it could be adapted to ceils from humans. Platelet aggregation is more sensitive to low concentrations of the mediator than is secretion and METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
