[75] Platelet aggregation and the influence of prostaglandins

642

BIOLOGICAL METHODS

[75]

subjected to various experimental treatments reflect stimulation of synthetic processes. In conclusion, the use of focused MW irradiation of the head of small laboratory animals, such as mice and rats, allows one to determine correctly endogenous levels of free PG-precursor fatty acids (e.g., FAA) and their oxygenated metabolites in brain regions and discrete nuclei, both in basal conditions (when they are very low) or after experimental treatments such as administration of centrally stimulating drugs, and hypoxia (when they are markedly increased).

[75] Platelet Aggregation and the Influence Prostaglandins

of

By JON M. GERRARD Platelets are blood cells with an important role in hemostasis. Aggregation is a quick and simple method for assessing their function in vitro. Since many prostaglandins and related compounds have important effects on platelet function, aggregation can be a useful tool in prostaglandin research. Its uses include (a) a bioassay of certain prostaglandins and thromboxanes; (b) the assay of platelet enzymes needed for synthesis of prostaglandins and thromboxanes; (c) the study of drugs that influence these processes; and (d) the evaluation of patient groups for alterations in platelet prostaglandin and thromboxane production. The use of aggregometry for these studies and its limitations will be discussed. Preparation of Platelet-Rich and Platelet-Poor Plasma Plasticware (polypropylene or polycarbonate) or siliconized glassware is used throughout. Platelet-rich plasma (PRP) is prepared following anticoagulation of blood using citrate or heparin. While several different formulations of citrate can be used, we use 93 mM sodium citrate, 7 mM citric acid, 0.14M dextrose, pH 6.5, and mix 1 volume of this anticoagulant with 9 volumes of blood. In rare instances where it is desired to compare the function of platelets in PRP from individuals with very different hematocrits, the citrate concentration must be adjusted to account for changes in hematocrit. 1 The anticoagulated blood is centrifuged at 100 g for 20 min at room temperature to separate PRP. PRP can also be prepared by using shorter centrifugation times with higher g forces, by keep1 j. G. Kelton, P. Powers, J. Julian, V. Boland, C. J. Carter, M. Gent, and J. Hirsh, Blood 56, 38 (1980).

METHODS IN ENZYMOLOGY, VOL. 86

Copyright © 1982by Academic Press, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181986-8

[75]

PROSTAGLANDINS VS PLATELET AGGREGATION

643

ing the product of the g force in the middle of the tube times the time in minutes between 1750 and 2400 (i.e., 200 g x 10 min = 2000). In some individuals a second shorter centrifugation (120 g for 5 min) may be nece ssary to eliminate a small degree of red blood cell contamination. In individuals with a platelet count of less than 50,000/~1 a slower spin is desirable (70 g for 20 min). For animal blood samples, the conditions of centrifugation required may vary with the species. Platelet-poor plasma (PPP) is prepared by centrifugation of anticoagulated blood or PRP at 5000 g for 10 min at 4 °. For comparison of different patient groups, it is wise to standardize platelet counts at 200,000//~1 or 300,000//d by diluting PRP with PPP. P r e p a r a t i o n of W as he d Platelets For many purposes (i.e., biochemical studies, evaluation of plasma factors, alteration or standardization of albumin concentration) washed platelets are desirable. The novice should be aware that there is " a r t " as well as science involved in washing platelets, and success depends on careful handling of these cells at all steps. Akkerman e t al. 2 have evaluated several washing procedures and found gel filtration to give the most metabolically and functionally intact platelets. However, they found considerable variation from one sample to another within a given technique. The citrate wash technique described below was not evaluated by Akkero man, but has been found in my laboratory regularly to give superb platelets as evaluated morphologically and functionally. In some hands, the EDTA wash, a procedure widely used in prostaglandin research, may be as good, though my experience coincides with that of Akkerman that such platelets tend to have more pseudopods and be functionally less intact. EDTA has a much higher affinity for calcium and magnesium than citrate and may leach these important cations from the membrane in addition to chelating them in the medium, leading to a mild to severe platelet injury that may be irreversible in some circumstances (note that even short exposure of platelets to EDTA at 37° produces irreversible changes3). Nevertheless, EDTA-washed platelets may be adequate for and useful in the study of arachidonic acid metabolism and the preparation of platelets for certain enzyme assays. The albumin wash technique, 4 has been used primarily to study platelet procoagulant activity. As it has not been used much in prostaglandin research, it will not be discussed here. 2 j. W. N. Akkerman, M. H. M. Doucet-de-Bruine,G. Gorter, S. de Graaf, S. Hefne, J. P. M. Lips, A. Numeuer, and J. Over, Thromb. Haernostasis 39, 146 (1978). 3 M. B. Zucker and R. A. Grant, Blood 52, 505 (1978). 4 p. N. Walsh, D. C. B. Mills, and J. G. White, Br. J. Haematol. 36, 281 (1977).

644

BIOLOGICAL METHODS

[75]

Centrifugation Methods For all centrifugation methods, the centrifugation speed should be the lowest that will pellet the platelets. This speed will vary depending on the density of the medium from 1000 g for 10 min in plasma to 300 g for 10 min in buffers with no albumin or plasma. The use of higher g forces for longer times will make the platelets harder to resuspend and may cause platelet injury. Citrate Wash. Add 1 ml of citrate anticoagulant (93 mM sodium citrate, 7 mM citric acid, 0.14 M dextrose, pH 6.5) per milliliter of PRP, chill to 4° for 5 min, then centrifuge at 1000 g for 10 min. Resuspend in one-tenth the original volume of the desired buffer by sucking the cells gently in and out through the tip of a plastic or siliconized glass Pasteur pipette and then make up to the desired volume. We routinely resuspend in Hank's balanced salt solution (HBSS), pH 7.4 (136 mM NaCI, 5.4 mM KCI, 0.44 mM KHzPO4, 0.34 mM Na~HPO4"7 H20, 5.6 mM D-glucose) either with or without albumin depending on the desired use of the platelets. For studies using ADP, epinephrine, or arachidonyl monoglyceride aggregation, we resuspend in HBSS containing 5-10% platelet-poor plasma. Where more than one wash in necessary (i.e., to remove the remaining plasma for thrombin aggregation) or desired, the once-washed platelet suspension is diluted 1:0.5 with citrate anticoagulant, centrifuged, and resuspended as above. Platelets should be incubated for 5 15 min at 37° to restore discoid shape and functional properties before performing aggregometry studies. An alternative citrate wash procedure has been described by Mustard) PRP is prepared from blood drawn into acid citrate dextrose (85 mM trisodium citrate, 65 mM citric acid, 110 mM D-glucose) (1 volume of anticoagulant to 6 volumes of blood) and then pelleted directly at 37°. Details of the resuspending buffers can be found in the original description of the method. EDTA Wash. Chill PRP to 4 °, add EDTA to a final concentration of 7.7 mM, and centrifuge at 1000 g for 10 min at 4°. Resuspend in the desired buffer. This washing procedure can be repeated several times as with the citrate wash. Baenziger and Majerus 6 advise using room temperature instead of 4 ° . Experience in my laboratory suggests that 4 ° is usually more satisfactory. Baenziger and Majerus also advise against initial resuspension in Tris-saline buffer as spontaneous aggregation may occur, though such buffers can be used after a second wash. The washed 5 j. F. Mustard, D. W. Perry, N. G. Ardlie, and M. A. Packham, Br. J. H a e m a t o l . 22, 193 (1972). N. L. Baenziger and P. W. Majerus, this series, Vol. 31, p. 149.

[75]

PROSTAGLANDINS VS PLATELET AGGREGATION

645

platelets can be kept at 4° until just before the aggregation studies, when they should be rewarmed to 37°. Gel-filtered platelets. Gel-filtered platelets are prepared by applying citrated PRP to a Sepharose 2B column (Lages et al. 7 recommend first thoroughly washing the Sepharose 2B with acetone and 0.9% NaCI) prepared after deaeration of the gel. The column is equilibrated with buffer, then the platelets are applied. The size of the column depends on the amount of PRP to be applied and the degree of separation of platelets from the plasma proteins. (Usually the gel volume should be 5-10 times the plasma volume for good separation.) The platelets, which are eluted at the void volume, are collected by visual observation of the change in opacity of the column effluent. Data of Akkerman et al.2 suggest that for most purposes CaZ+-free Tyrode solution (137 mM NaCI, 2.68 mM KC1, 0.42 mM NaH2PO4, 1.7 mM MgCI2, 11.9 mM NaHCOa, 5.6 mM D-glucose, 2 g of bovine albumin per liter, pH 7.3) is an excellent buffer to use although other buffers also may be satisfactory. Choice of Resuspending Buffer Resuspend platelets at 200,000 to 500,000/~1 for optimum studies of platelet aggregation. In choosing a resuspending buffer, be aware of several factors. Once separated from plasma, washed platelets tend to become rapidly depleted in ATP unless glucose (usually 5.6 mM) is added to the resuspending medium. Some potassium should also be included in washing and resuspension buffers since platelet levels may become depleted during washing. Apyrase is frequently added to degrade any ADP released into the plasma during washing procedures, since such ADP can make the platelets refractory to further ADP stimulation. Heparin is sometimes added to the first resuspension buffer to prevent effects of small amounts of thrombin that may be generated. For investigators interested in prostaglandins, one of the most important considerations in choosing a resuspending buffer relates to the albumin concentration. Albumin binds arachidonic acid, and the concentration of this fatty acid required to produce aggregation is thus critically dependent on the albumin level of the medium. In plasma, the dose of arachidonic acid needed to produce aggregation varies from 0.2 to 1.5 mM depending on the intrinsic sensitivity of the platelets used. In contrast, platelets washed three times with the EDTA wash to remove plasma and then resuspended in HBSS, pH 7.4, aggregate using 0.3-5 pA4 arachidonic acid, a concentration approximately 1000 times lower. 8 If too high a concentration of arachidonic 7 B. Lages, M. C. Scrutton, and H. Holmsen, J. Lab. Clin. Med. 85, 811 (1975). 8 j. M. Gerrard, J. C. Peller, T. P. Krick, and J. G. White, Prostaglandins 14, 39 (1977).

646

BIOLOGICAL METHODS

[75]

acid is used relative to the concentration of the albumin, platelet lysis results. Such lysis may occur with as little as 20/zM arachidonic acid in the absence of any albumin. Lysis has been mistaken for aggregation by some investigators. Where it occurs it is not inhibitable by aspirin. If aspirin pretreatment of the platelets (100/zM for 15 min) does not completely block arachidonic acid aggregation, the fatty acid is probably causing cell lysis. The concentration of albumin also has an effect on the dose of prostaglandin endoperoxides and A23187 (a calcium ionophore), though to a lesser extent than with arachidonic acid. Since albumin can enhance the conversion of PGH2 to PGD~ this consideration is important to studies of prostaglandin endoperoxides and their metabolism by platelets. For most aggregating agents, fibrinogen may be necessary for the aggregation. Since platelet alpha granules contain fibrinogen, it is not essential to add fibrinogen with an agent that causes granule secretion, but added fibrinogen is needed for ADP aggregation under circumstances where no secretion is produced. For ADP or epinephrine aggregation of citrate-washed platelets, these cells can be resuspended in HBSS containing 5-10% PPP, or in some circumstances in buffers containing albumin and fibrinogen? These washed platelets aggregate at similar concentrations of these agents to those used in native plasma. Fibrinogen used for this purpose may need to be treated with diisopropylfluorophosphate to avoid any contribution of procoagulant material contaminating the protein preparation.a External calcium or magnesium are also essential for platelet aggregation. Since albumin binds calcium, the concentration needed may vary with the concentration of albumin. Changing the calcium level has important effects on the second wave of platelet aggregation and on the release of arachidonic acid from platelet phospholipids, the second-wave aggregation and arachidonic acid release being suppressed at higher calcium concentrations 9,10 For arachidonyl monoglyceride, we routinely use citrate-washed platelets resuspended in 10% plasma, as we have found these conditions to be optimal, perhaps relating in part to the optimum conditions for delivering arachidonyl monoglyceride in liposomes to platelets, n Platelet Aggregation The platelet aggregometer measures the transmission of light through a stirred cuvette maintained at a constant temperature, usually 37° (Fig. 9 j. F. Mustard, D. W. Perry, R. L. Kinlough-Rathbone, and M. A. Packham, Am. J. Physiol. 228, 175 (1975). 1o M. J. Stuart, J. M. Gerrard, and J. G. White, Blood 55, 418 (1980). 11 j. M. Gerrard and G. Graft, Prostaglandins Med. 4, 419 (1980).

[75]

PROSTAGLANDINS VS PLATELET AGGREGATION

647

f i~iii~iii~i~iiii~i ~ii~iii~iii~

lib

!

TRANsuis

tA

Fla. 1. The platelet aggregometer evaluated platelet function by its ability to measure light transmission through a suspension of stirred platelets. As shown in the tracing on the right, the initial suspension is opaque and the resting discoid platelets give baseline oscillations. As aggregation proceeds, platelets form larger and larger clumps and the solution clears until maximum aggregation is achieved.

1). The upper and lower limits of the recording pen are first set using the suspending medium without the platelets (PPP or washed platelet resuspending buffer) as 100% aggregation, and the platelet suspension (PRP or washed platelets) as 0% aggregation. In Fig. 1 a sample aggregation tracing is shown to facilitate understanding of the test and its uses. At the bottom of Fig. l, light is transmitted through a stirred relatively opaque solution of platelets (0% aggregation). The birefringence of stirred resting platelets that are discoid in shape produces oscillations in the light transmission as recorded at the right. In the example shown, following addition of an agonist at A, there is first a change in the baseline oscillations to give a straight line. The initial loss of birefringence results from a change in platelet shape to a more spherical form with pseudopods. Subsequent to this, the extent of light transmission increases to the extent that platelets clump together into aggregates. When all platelets are clumped into a few large aggregates, the remainder of the solution is clear (100% aggregation). Since the assay conditions depend critically on measurement of light transmission, it is very important to be aware of and to allow for changes in light transmission resulting from addition of the agonist solution if careful quantitation is desired. Since platelet aggregation responses may vary with time, and since

648

BIOLOGICAL METHODS

[75]

spontaneous aggregation may occur under some circumstances, it is always essential to add a buffer control, and in evaluating the effects of inhibitors, such controls should be run at the beginning and the end of an experiment. Buffers and Solvents for Use with Specific Agonists In general, water-soluble reagents can be made up satisfactorily in HBSS or Tris-buffered saline. For compounds that are not readily soluble in water, two organic solvents that are water miscible are frequently used --ethanol and dimethyl sulfoxide. Both of these are inhibitory and/or toxic to platelets in high concentrations. The final concentration of either after addition to the platelet suspension should be kept less than 0.5% whenever possible and must never be more than 1%. Liposomes have been found useful to deliver arachidonyl monoglyceride to platelets and may be useful for other agents in the future. Epinephrine (as the chloride salt), ADP, and thrombin are all water soluble and pose no particular problem in reconstituting in HBSS or Trisbuffered saline. Collagen can be prepared as a suspension or, as acid-soluble collagen, can be solubilized in a dilute solution of acetic acid. A23187 is soluble in dimethyl sulfoxide or ethanol and can be added in a few microliters to the platelet suspension. Lysophosphatidic acid is soluble in 50% ethanol, or it can be used as a suspension in an aqueous buffer such as HBSS. Arachidonic acid can be used as the sodium, potassium, or ammonium salt in an alkaline buffer (i.e., 0.1 M Tris, pH 8.5) in an ethanolcarbonate solution, or can be added dispersed in a plasma or albumin solution. For PGG~, and PGH2, we have routinely evaporated the solvent under N2 and then added the platelet suspension immediately. Thromboxane A2 can be prepared on addition of prostaglandin endoperoxides to a preparation containing thromboxane synthase. Arachidonyl monoglyceride needs to be made up in liposomes. Liposomes made from beef heart phosphatidylcholine or from phosphatidylinositol from yeast or soybeans are satisfactory. Liposomes can be made satisfactorily by sonication of 20 mM phospholipid with 8 mM arachidonyl monoglyceride in distilled water using either a Bronson probe sonicator (3 x 10 sec at 4°) or a bath-type sonicator (15 min at 37°)) 1

Analysis of Aggregation Patterns Understanding patterns of platelet aggregation with various agents and conditions is essential to use of the aggregometer. In this section, emphasis will be placed on changes in patterns with the type or dose of agonist. Use of platelet aggregometery in a quantitative or semiquantitative

[75]

PROSTAGLANDINS VS PLATELET AGGREGATION

649

fashion requires attention to changes with dose in order to choose a parameter to measure to evaluate greater or lesser degrees of aggregation. Patterns of platelet aggregation of particular relevance to prostaglandin research can be grouped into three categories as shown in Fig. 2. Epinephrine, the agonist shown in pattern 1 of this figure is unique in that it causes a first wave of aggregation in which platelets clump together with little or no change in shape. No granule secretion or synthesis of prostaglandins occurs during the first wave of epinephrine aggregation. During the second wave of epinephrine-induced aggregation, shape change does occur together with the synthesis of prostaglandins and thromboxanes and the secretion of platelet granule contents. Studies with inhibitors and congenitally deficient platelets suggest that in most circumstances some degree of both thromboxane synthesis and secretion are necessary for this second wave. A number of agonists, including ADP, thrombin, lysophosphatidic acid, prostaglandin endoperoxides, and thromboxane As, when added to platelets in PRP produce pattern 2 of platelet aggregation as shown in Fig. 2. Low doses of agonist cause shape change only, whereas slightly higher

2

•

SEC,TxA2

11~3RF_A,SU~IGAGONIST CONCENTRATION

FIG. 2. Three types of aggregation patterns important for investigators studying prostaglandins are shown (see text). For each type the change in pattern with increasing dose of agonist is shown so that the reader can appreciate how the curves change with dose. Quantitation of aggregation depends on the use of a parameter that will vary with dose. The choice of parameter varies with the pattern of aggregation seen, as discussed in the text. In some circumstances, there are two waves of aggregation. Where this occurs, endogenous thromboxane A~ (TxA~) production and secretion (SEC) are associated with the second wave of aggregation. Inhibitors of the prostaglandin endoperoxide synthase, such as aspirin and indomethacin, will prevent the development of the second wave of aggregation under such circumstances.

650

BIOLOGICAL METHODS

[75]

doses of agonist produce a reversible first wave. Still higher doses produce a biphasic response with two waves of aggregation, and at the highest doses only a single complete wave of aggregation is seen. The intermediate form in which there are two waves is less prominent with platelets from certain individuals and with some agents, though it may occur with all these agents in PRP or in washed platelet preparations in which there is substantial plasma or albumin. In general, the extent to which a second wave is obtainable with ADP and with the other agents depends on the concentration of calcium with a greater likelihood of a second wave response at a lower calcium concentration (i.e., Mustard et al. 9 showed a second wave and secretion with ADP stirred with washed platelets in 1 mM Mg and no Ca, but no second wave or secretion with 1 mM Mg and 2 mM Ca). The third pattern of aggregation shown occurs with collagen, arachidonic acid, and A23187 in platelet-rich plasma, with arachidonyl monoglyceride (AMG) added to platelets in 10% plasma, and with collagen, arachidonic acid, prostaglandin endoperoxides, and thromboxane A2 added to platelets washed and resuspended in the absence of plasma. Rarely A23187 or AMG have been seen or reported to produce a double wave when added to PRP as shown in pattern 2. In many circumstances, aggregation tends to be an all-or-none phenomenon as shown in pattern 3 of Fig. 2, with the major change with increasing agonist concentration above threshold levels being in the time to onset of aggregation or the slope of aggregation. In some circumstances, particularly with washed platelets, aggregation may progress in incremented steps (i.e., 20% aggregation at a lower dose, 40% aggregation at a higher dose, and 100% aggregation at the highest dose (each having a single irreversible wave). Q u a n t i f i c a t i o n of Platelet Aggregation

Platelet aggregation can be used in a quantitative or semiquantitative fashion to analyze various questions. For most purposes, it is desirable to choose a parameter of the aggregation response that changes in a linear fashion proportional to the dose or the log of the dose of the agonist. When this does occur it is usually only in a relatively limited dose range. Use of platelet aggregation in a quantitative fashion relies on establishing this dose range and the response. A problem is that each individual's platelets may respond a little differently, so that a dose response in one individual may be quite different from that in another. For this reason, comparisons between different individuals is often done using the dose to achieve a specific effect (i.e., 50% aggregation). The variation in response also means each platelet preparation must be tested for its dose response before using an inhibitor (i.e., PGI2) if it is desired to quantitate the amount of agonist or inhibitor.

[75]

PROSTAGLANDINS VS PLATELET AGGREGATION

651

In studying inhibitors, there is a danger in using too high or too low a dose of agonist (i.e., if the initial dose response shows that 2/~M ADP gives 20% aggregation, 4/zM ADP gives 50% aggregation, 6 pA/ ADP gives 80% aggregation, 8/.tM ADP gives 90% aggregation, 10/zM ADP gives 95% aggregation, and 30/~M gives 96% aggregation, then the appropriate dose to check the effect of an inhibitor is 4 - 6 pA/ADP. If 30/zM ADP is used, considerable inhibition could be achieved with very little change in the aggregation pattern. The parameter to be used in assessing changes in aggregation with agonist concentration varies with the type of agonist. For agonists in pattern 1 (Fig. 2), the time to 50% aggregation, or the area under the aggregation curve up to 3 or 5 rain tends to be most useful. For agonists with pattern 2, the height (percentage aggregation) of the first wave or the area under the aggregation curve is useful. For agonists with pattern 3, the time to 50% aggregation, the slope of the aggregation curve, or the area under the aggregation curve is usually most rewarding; however, in some circumstances the percentage of aggregation is useful. Measurements of granule secretion may be more accurate than evaluation of the aggregation tracing at high agonist doses. In general our experience is that except for aggregating agents in pattern 3, slope is not reliable, although some investigators have managed to achieve conditions where slope can be used with agonists of pattern 2. Some investigators have used the time to onset of aggregation or the time to 100% aggregation. Our experience is that a higher degree of accuracy can be achieved using the time to an intermediate degree of aggregation (usually 50%, but sometimes 10% or 90~), since the start and completion of aggregation as determined from an aggregation curve is somewhat arbitrary. With all quantitative uses of platelet aggregation, attention needs to be given to keeping the preparation of platelets stable in their response for some length of time. In PRP, platelets usually produce a rapid rise in pH that will alter aggregation patterns. The rise in pH can be avoided by replacing the air in the tube with a CO2/O2 mixture and stoppering the tube. For additional steps to reduce variability in platelet aggregation, the investigator should consult Newhouse and Clark. 12 Platelet Aggregation as a Bioassay for Prostaglandin Endoperoxides and Thromboxane As Platelet aggregation can be used quantitatively as described above to detect prostaglandin endoperoxides and thromboxane As. As with other bioassays, it is usual to reduce interference with the assay from other 12p. Newhouse and C. Clark, in "Platelet Function" (D. A. Triplett, ed.), p. 63. American Society of ClinicalPathologists, Chicago, 1980.

652

BIOLOGICAL METHODS

[75]

agonists. As a minimum, most investigators have pretreated the platelets with aspirin or indomethacin to inhibit any platelet response to arachidonic acid. In some circumstances, it may be important to add heparin or hirudin to inhibit effects due to thrombin. Platelets can be made refractory to ADP, yet maintain their responsiveness to endoperoxides and thromboxane A~, by preincubating the platelet sample with ADP. 13 Some EDTA-washed platelet preparations are also refractory to ADP but respond well to endoperoxides. Some investigators have succeeded in using a combination of creatine phosphate and creatine phosphokinase to convert ADP to ATP to prepare a platelet suspension that responds nicely to endoperoxides and thromboxane A2 but does not respond to ADP. However, others have found that such preparations do not respond well to endoperoxides or thromboxane Az when ADP is inhibited. It is possible that some preparations of creatine phosphate or creatine phosphokinase have contaminants that inhibit aggregation to endoperoxides. However, given the current controversy, this methodology cannot be recommended. Platelet Aggregation as a Bioassay for PGI~, PGD2, and PGE1 These three prostaglandins can be assayed owing to their inhibitory effects on platelet aggregation. Bioassays have been devised in which a standard dose of agonist (usually ADP) is added to PRP (as explained above this dose should be one that gives 50-80% aggregation). The dose of PG needed to give various degrees of inhibition is then established by adding the PG in varying concentrations at a fixed time (usually 30 sec) before addition of ADP on the aggregometer. The solution containing an unknown concentration of PG can then be added in a similar fashion and its concentration determined by comparison with the effect of the calibrating doses. In these experiments it is important to recalibrate the platelet response at intervals during a long experiment and both before and after a short experiment to check that the platelet response to ADP, and its inhibition by PG, has not changed substantially during the assay. The assay is not selective and will detect any inhibitory PG or indeed other inhibitor. An approach to identify a selective effect of PGI2 has been to use the short half-life of PGI2 in neutral or acid aqueous solutions to show that the effect of PGI~ in the test sample behaves like PGI2 in this respect.

Assay of Platelet E n z y m e s In general, aggregation can be used only as a quick, crude evaluation for the presence, absence, or substantial change in activity of an enzyme. Further biochemical quantitation is an essential subsequent step. la j. M. Gerrard and J. G. White, Prog. Hemostasis Thromb. 4, 87 (1978).

[75]

PROSTAGLANDINS VS PLATELET AGGREGATION

653

Prostaglandin Endoperoxide Synthetase (Cyclooxygenase). Add arachidonic acid to PRP or washed platelets and follow aggregation. A response to arachidonic acid in plasma or in washed platelets indicates that the enzyme is present. With washed platelets, the aggregation should always be retested with aspirin-treated samples to make sure this abolishes the response (see earlier discussion regarding lysis). The platelets should also be tested with an endoperoxide or endoperoxide analog to be sure that the cells respond to these compounds and do not have another defect. Variation in the inherent responsiveness of platelets unrelated to the activity of this enzyme may make comparisons of enzyme present in different individuals difficult, although an enhanced responsiveness of diabetics to arachidonic acid has beed used to indicate an enhanced enzyme activity. TM In view of the major effect of albumin on the platelet response to arachidonic acid, it may be desireable to standardize the amount of albumin in the platelet suspension. Thromboxane Synthase. Gorman et al. is have used platelets in a fashion to detect abnormal thromboxane synthase activity (no response to PGH2, but normal response to a thromboxane analog,) but their interpretation has been questioned. TM Since thromboxane A2 is more active than PGH2, platelet microsomes containing thromboxane synthase when mixed with PGG2 or PGH2 produce considerably more aggregation than PGG2 or PGH2 alone, and this difference could be used to evaluate this enzyme. ~7 At present it seems wise to perform another assay of this enzyme in addition to platelet aggregation. Diglyceride Lipase. Add arachidonyl monoglyceride to washed platelets in 10% plasma. Aggregation by this agent is believed to require this lipase, and the presence of aggregation to arachidonyl monoglyceride implies that this enzyme is present. If there is no aggregation, aggregation to arachidonic acid should be evaluated to check whether subsequent conversion and the response to arachidonic acid are intact. Phospholipase C and A2. There is currently still debate as to the precise events involved in release of arachidonic acid from platelet phosholipids, and until this is resolved only a few comments can be made. Second wave aggregation to epinephrine or ADP which can be blocked by pretreating the platelets with aspirin, implies that the platelets are capable of releasing arachidonic acid from their phospholipids, converting it to thromboxane Az, and responding to this agent. Absence of such a second 14 p. V. Halushka, D. Lurie, and J. A. Colwell, N. Engl. J. Med. 297, 1306 (1977). 1~ R. R. Gorman, G. L. Bundy, D. C. Peterson, F. F. Sun, O. V. Miller, and F. A. Fitzpatrick, Proc. Natl. Acad. Sci. U. S. A. 74, 4007 (1977). 16 D. E. Maclntyre, in "Platelets in Biology and Pathology" (J. L. Gordon, ed.), Vol. 2, p. 211. Elsevier/North-Holland, Amsterdam, 1981. 17 S. Moncada, P. Needleman, S. Bunting, and J. R. Vane, Prostaglandins 12, 323 (1976).

654

BIOLOGICAL METHODS

[75]

wave in the presence of a normal response to arachidonic acid could be associated with defective release of arachidonic acid, but may also reflect defective platelet granule content or decreased metabolic ATP. T h e Evaluation of Drugs Two basic questions may be asked: What is the mechanism of action of an inhibitory drug; arid What is its potency? To answer the first question, it is important to compare the pattern of inhibitory effects seen with that produced by known inhibitors. Agents that raise cyclic AMP, including PGI2, have a general depressant action on aggregation by all agents (i.e., first-wave aggregation as much as second-wave aggregation), although at low concentrations there may be selective inhibition of arachidonic acid aggregation compared to inhibition of thrombin, collagen, or ADP. TM Agents that inhibit the prostaglandin endoperoxide synthase enzyme inhibit second-wave epinephrine or ADP aggregation and inhibit aggregation by arachidonic acid, but not aggregation due to endoperoxides. A selective diglyceride lipase inhibitor should inhibit aggregation due to arachidonyl monoglyceride, but not aggregation produced by arachidonic acid. A selective thromboxane receptor antagonist inhibits aggregation produced by thromboxane, but not aggregation due to ADP. To assess the potency of a drug, the conditions are similar to those used to quantitate inhibitory PGs described earlier. However, for some circumstances, it is desirable to use an aggregating agent other than ADP. For an agent that inhibits the prostaglandin endoperoxide synthase, arachidonic acid aggregation or second-wave epinephrine aggregation are more useful than ADP aggregation, sirlce ADP is less affected by endoperoxide synthase inhibitors. T h e Study of Patient Groups In comparing samples from different inidividuals, it becomes important to standardize platelet counts, to be particularly careful about good venipuncture technique and red cell contamination of samples, and to use a standard time interval after drawing the blood for the test. Variation in any of the above can influence results. Acknowledgments This work was supported in part by MRC Grant 7396, by a grantfrom the NationalCancer Institute of Canada, and by an MRC Scholarship. x8G. H. R. Rao, K. R. Reddy, and J. G. White,Prostaglandins Med. 6, 75 (1981).