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Characterization of the platelet response to exogenous arachidonic acid
THROMBOSIS RESEARCH 22; 157-166, 1981 0049-3848/81/010157-10$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
CHARACTERIZATION OF THE PLATELET RESPONSE TO EXOGENOUS ARACHIDONIC ACID
Joseph C. Fratantoni and Betty J. Poindexter
Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland (Received 15.1.1981; in revised form 7.4.1981. Accepted by Editor J.S. Finlayson) ABSTRACT
When aggregation and release are the end points, washed human platelets treated with a range of concentrations of arachidonic acid display a bell-shaped dose-response curve. The peak is at approximately 200 PM, followed by a sharp decrease with minimum reaction at 400 uM. Prostaglandin synthesis, measured by malondialdehyde production, reaches peak activity at 400-500 uM and does not decrease. Platelet lysis, as manifested by lactate dehydrogenase release, increases only in response to arachidonate concentrations above 400 yM. When platelets are treated with 400 nM arachidonate for three minutes, subsequent reaction with thrombin, but not with A23187, is inhibited. This inhibitory activity is not sensitive to aspirin, indomethacin or ETYA and cannot be transferred in the supernatant fraction of the platelet suspension. The inhibitory activity generated from platelets reacted with 400 pM arachidonate is not a product of the cyclooxygenase or lipoxygenase systems.
INTRODUCTION The metabolic conversion of arachidonic acid to the prostaglandins and thromboxanes is causally associated with platelet aggregation and release reactions (1,Z). This pathway is physiologically significant since arachidonic acid is derived from membrane phospholipids via enzyme reactions which appear to be of fundamental importance in platelet activation. Several investigators have observed that addition of exogenous arachidonic acid to platelet preparations results in platelet reactions (3-6). Only recently, however, has the relationship between increasing concentration of arachidonic acid and platelet response been reported (7,8). When platelet release or aggregation is the measured reaction, addition of arachidonic acid (in the form of sodium arachidonate) yields a bell-shaped dose-response curve, that is, a biphasic response composed first of increasing, then of decreasing reaction intensity. Key Words:
Arachidonic Acid, Platelet Aggregation, Prostaglandins. 157
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In the course of studying the response of stored platelets to various stimuli, we have observed the biphasic relationship between aggregation or release, and arachidonate concentration. We have further noted that prostaglandin synthesis -_ per se, as indicated by malondialdehyde (MDA) production, does not demonstrate a biphasic response to progressive arachidonate levels. In this report, the following questions have been addressed regarding these observations: 1) Is the biphasic response the result of platelet lysis due to the detergent effect of sodium arachidonate? 2) Platelets exposed to higher concentrations of arachidonate display decreased reactions. Are such platelets similarly less reactive to other stimuli? 3) Is the altered platelet reactivity observed the result of metabolic products of arachidonate? MATERIALS AND _--METHODS Sample preparation Washed platelets (WP) were prepared by a modification of the method of Packham et al. (9). Blood from healthy donors who had ingested no known platelet-reactive medication for 10 days, except as noted, was collected into l/7 volume of citrate phosphate dextrose anticoagulant solution (CPD). Aspirin-treated platelets were obtained by administering 600 mg of aspirin to the donor two to four hours prior to venipuncture. Informed consent was obtained from each subject prior to the initiation of studies in accordance with the requirements of the Clinical Research Colrmittee. Platelet rich plasma (PRP) was separated at 200 x g for 15 min. [14C]serotonin (10'6w) and heparin (25 units/ml) were added and the mixture incubated for 30 minutes at 37 OC. The pH was adjusted to 6.3-6.5 with 0.25 M citric acid. The platelets were separated by centrifugation at 900 x g for 10 minutes and washed twice with Tyrode's buffer containing magnesium (1 mM), calcium (2 mM), apyrase (3-5 u/ml), and albumin (0.35 g/100 ml). Platelets were finally suspended in Tyrode's buffer, pH 7.4, containing albumin, magnesium, and calcium, as above, and apyraee (0.3-0.5 u/ml). WP were kept at 37 OC until used. All operations were done in polypropylene tubes (Falcon No. 2059 or 2063). Platelet aggregation Class cuvettes-and metal stir bars were siliconized. The aggregation response of 1 ml WP was observed in a Payton dual channel aggregation module (Payton Associates, Inc., Buffalo, NY). Aggregation was allowed to proceed for three minutes, at which time aliquots were removed and assayed as described below. Assay for released [14C]Serotonin --0.2 ml of reacted WP was added to 0.5 ml 0.13 M formaldehyde, with mixing. The mixtures were centrifuged at 2500 x g, for 10 minutes and the supernatant fluid counted in a Beckman liquid scintillation counter. Release due to factors other than arachidonic acid were accounted for with a saline control. A sample of labelled WP which had been lysed by mixing 1:lO with water was counted and used as the 100% value for calculation of percent release. Assay for MDA Production 0.7 ml of reactemP was taken from the cuvette, mixed into 0.7 ml 20%
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trichloroacetic acid in 0.6 M NC1 and assayed for MDA according to the method of Smith et al. (10). Tyrode's solution was used in place of platelet poor plasma for the controls. Difference spectra were performed on a Gary Model 219 spectrophotometer and A532 recorded. Lactate dehydrogenase Reagents and procedures from Sigma (LDH assay kit 340-UV) were used to perform a modification of the method of Wrobelewski et al. (11). The assay was standardized with Fisher Normal and Abnormal Clinical Chemistry Control Sera. Reagents Arachidonic acid, sodium salt was obtained from Nu-check, Elysian, MN, [14C]serotonin binoxalate from New England Nuclear Corp., and PentexB human albumin, fraction V, from Miles Laboratories. Apyrase was prepared from potatoes following the method of Molnar and Lorand (12), assayed according to Niewiarowski (13) and stored at -40 OC. Human thrombin, lot H-l, was provided by Dr. David Aronson, Bureau of Biologics. Eicosatetraynoic Acid (ETYA) was kindly provided by Dr. W. E. Scott, Hoffman-La Roche, Inc., Nutley, NJ).
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FINAL ARACHIDONATE CONC. (@VI)
FIG. 1 Aggregation (0 -UJ), 114C]serotonin release (0 -0) and MDA production (A-A) response of washed platelets to sodium arachidonate. Values displayed are means of nine experiments using seven donors.
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RESULTS -Demonstration of the Biphasic Dose Response Curve Simultaneous measurement of aggregation, serotonin release, and MDA production in response to 0 to 400-cM arachidonic acid is shown in Figure 1. Al.1 donors tested demonstrated biphasic aggregation and release curves. Blood from a donor who had ingested 600 mg aspirin 12 hours prior to venlpuncture yielded platelets which were non-reactive to arachidonic acid at concentrations up to 400 PM. At arachidonate concentrations above 400 pM, serotonin was released at levels above 50% of total platelet radioactivity. Aggregation response to these concentrations varied from shape change alone to transmission readings above 60%. MDA production appeared to be maximal at 400 to 500 uM and did not change at higher concentrations. The release response to arachidonate levels >400 ufl was not decreased by prior ingestion of aspirin. The results of lactate dehydrogenase (LDH) asBay of the supernatant fractions of washed platelet suspensions are shown in Table 1. Platelet counts on suspensions of WP treated with 50-800 PM arachidonate showed no changes before and after When examined with phase microscopy, addition. it was noted that platelets treated with 600 PM arachidonate had lost their discoid shape and had become spherical. TABLE 1 LDH in Suuernatant Fraction of Platelet Susnensions St irked with Arachidonate for 3 Minute;. Final Concentration of Arachidonate CUM)
0
100 200 300 400 500 600 800
LDH (X of total in suspension + S.E.M)*
1.83 3.23 3.89 4.86 3.95 7.81 12.21 20.927
+ 7 + ? T T 7 -
1.2 1.8 0.9 1.3 1.2 1.0 1.7 1.5
* Total LDH in suspension determined by assay on sonicated samples which yielded a mean value of 645.5 international units/l012 platelets. Each value is the average of 4 or 5 assays.
These observations support the high concentrations of arachidonate specific lysis, and further suggest intact at these concentrations.
conclusions of Linder, et al. (7), that (in our system >400 PM) result In nonthat prostaglandin synthesis remains
Mechanism of the Biphasic Response Characzrization of the decreased aggregation response to high arachidonate levels was explored by reacting UP with 400 uM arachidonate then testing reactivity of the platelet suspension to thrombin. Figure shows the aggregation curves resulting from treatment of normal WP with
and 2A
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40 $1 or 400 pM arachidonate, followed after three minutes by addition of thrombin. The thrombin concentration required to achieve maximum aggregation after 400 nM arachidonate is 10 times that required after exposure to 40 nM arachidonate. A control (prior addition of buffer only) shows approximately the same reactivity to thrombin as the 40 pM arachidonate pretreatment sample.
.16 u!ml .12 u!mt .oBulml
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FIG. 2 Aggregation of washed platelets stirred with indicated concentrations of arachidonate (A) followed by thrombin (T). The interval between arachidonate and thrombin additions was three minutes. Results from one of six similar experiments. A: Normal platelets; B: Aspirin-treated platelets.
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In order to test the effects, on inhibition, of blocking the cyclooxygenase pathway inhibitors of this enzyme were added to the system. Figure 2B shows results of an experiment using WP derived from a donor who had ingested 600 mg of aspirin two hours prior to venipuncture. These platelets did not aggregate in response to 200 pM arachidonate. Other with similarly treated platelets, demonstrated that such cells experiments, or MDA responses to arachidonate at did not yield release, aggregation, concentrations from SO-400 pM. However , the inhibition of thrombin reactivity induced by pretreatment with 400 PM arachidonate is clearly present. The thrombin concentration required to stimulate maximum aggregation after 400 pM arachidonate pretreatment is approximately five times that required after 40 pM arachidonate pretreatment. The variability noted with other donors and the inherent problems of aggregation end points do not permit us to conclude from these data whether or not there is any difference between inhibition found with normal vs. aspirin-treated platelets. Addition of indomethacin in vitro, at a final concentration of 8 VU, similarly had no effect on the inhibition of thrombin reactions by e. 400 ~.IM arachidonat Arachidonate is also metabolized via the lipoxygenase system, which, in platelets, yields primarily a 12-hydroxylated derivative. This system can be inhibited by eicosatetraynoic acid (ETYA) at concentrations which do not fully inhibit the cyclooxygenase pathway (14). When 2.5 pM ETYA was added to WP, the amplitude of the aggregation response to 200 PM arachidonate was approximately one-half that of control WP. ETYA, at a final concentration of 2.5 nM was then added to a similar experimental system and the results are shown in Figure 3. The ETYA was added 1 minute prior to addition of arachidonate while a similar volume of solvent (DMSO) was added as a control. system did not differ from the control in any The ETYA treated way. Finally, the calcium ionophore A23187 was used as the test stimulant in place of thrombin. Concentrations from 0.1 to 5.0 PM A23187 gave progressively increasing aggregation and release responses which were not affected by prior addition of 400 pM arachidonate. In order to test for a soluble, extracellular inhibitor, WP from normal and aspirin-treated donors were reacted with either buffer, 40 PM arachidonate or 400 uM arachidonate for three minutes, then centrifuged at 900 x g, for 10 minutes, at 22 OC. The supernatants were mixed with an equal volume of WP derived from an aspirin-treated donor. Aspirin-inhibited platelets were used as the test system to avoid stimulatory effect of arachidonate (in the supernatant) on aggregation. The platelets were tested within 60 minutes of resuspension. Eleven such experiments were performed using platelets from nine subjects. The supernatant fractions from cells treated with either buffer or 40 IJM arachidonate, when mixed with test cells, reacted similarly with thrombin, showing aggregation >80% of maximum at threshold concentrations of 0.05 to 0.1 units of thrombiniml. The addition to test cells of supernatant fractions from platelets treated with 400 pM arachidonate gave more variable results. In two experiments, the buffer control threshold concentration was 0.06 units/ml, while the threshold with the 400 PM arachidonate supernatant was 0.1 unit/ml. In the remainder of the experiments, no consistent pattern of inhibition by supernatant from cells treated with 400 pM arachidonate was seen, even when these cell suspensions contained lOa platelets/ml. In order to test for the presence of activators in the supernatant fractions, 400 pM arachidonate was stirred for three minutes at 37 Oc with either normal platelets, aspirin-treated platelets or Tyrode’s buffer. An aliquot of supernatant from each mixture was added to an equal volume of
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washed normal platelets and aggregation and release measured. The solutions which had been in contact with platelets for three minutes, when added to test cells, gave 0 to 4% aggregation and 0 to 6.3% release. The Tprode's buffer control which contained a final arachidonate concentration of 200 uM after mixing with the test platelets, gave 57% aggregation and 48% release.
12
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FIG. 3 Aggregation of washed, aspirin-treated platelets with indicated concentration of arachidonate (A) and Thrombin (T). ETYA (E) was added as 10 microliters of a 0.25 mM solution in DMSO. *lO microliters of DMSO added to sample 3 at "E".
DISCUSSION -Addition of arachidonate to washed hrrmanplatelets results in measurable aggregation, release of intracellular serotonin and formation of MDA. In these studies we have observed that 1) the arachidonate dose-response curve is bell-shaped if aggregation or release are measured, but not when MDA production is the end point; 2) exposure of platelets to 400 pM arachidonate results in inhibition of subsequent reactivity to thrombin but not to the calcium ionophore A23187; 3) the inhibitory activity is not affected by prior treatment with aspirin, indomethacin or ETYA; 4) the inhibitory activity could not be demonstrated in the plateletarachidonate supernatant; and 5) the effects seen as a result of 400 PM arachidonate are not due to platelet lysis. The potency of arachidonate in this system is a function of the protein concentration in the reaction medium. In the final suspension buffer employed, containing 3.5 g of albumin per liter, the nadir of platelet reactivity at 400 uM is consistent with previous reports, using either low albumin buffers or plasma (7). In our hands, this inhibition is not affected by aspirin or indomethacin. Clemmons, et al. (8) report that their inhibitor is aspirin sensitive, but their system is quite different (dog platelets, PRP, serotonin stimulation) and the limited data available do not permit further explanation.
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The determination of LDH in the supernatant fraction of WP treated with arachidonate clearly demonstrates that cell lysis is not a factor with arachidonate concentrations <400 w. Kinlough-Rathbone et al. (15) have shown that measurable lysis accompanies the release reaction stimulated by Our data similarly show the lowest LDH levels in the thrambin or collagen. buffer control sample, but also show no differences in LDH from samples treated with all concentrations of arachidonate 5400 UM. Aa the concentrations of arachidonate are raised above 400 $f, increasing lysis The aspirin-insensitive phenomonena occurs manifested by higher LDH levels. observed with the aggregometer and [ 14C]serotonin procedures are thus most readily explained by cell lysis. Experiments which utilized the supernatant fractions from platelet suspensions treated with 400 IJMarachidonate did not clearly demonstrate a At first these aggregation results were not as soluble inhibitor. reproducible as other studies. Very careful attention to sample preparation (uniform platelet count, samples to be compared run simultaneously, identical mixing times for test and control samples) eliminated some of this variation. We believe additional problems result from constituents released from the As can be seen in Fig. 1, platelet upon treatment with 400 I.IMarachidonate. release is inhibited at that level of arachidonate but is not zero, and it varies with time and with the subject. Such factors may account for the observations by others (7,B) of a soluble inhibitor. Agents known to block arachidonate metabolism via the cyclooxygenase pathway (aspirin, indomethacin) or the lipoxygenase pathway (ETYA) did not interfere with the inhibition observed with 400 $i arachidonate. MDA production is not inhibited, and is evidence that arachldonate is Therefore, the metabolized to products of the cyclooxygenase pathway. platelet must be prevented from undergoing thrombin-stimulated aggregation and release reactions by its interaction with arachidonate itself. The ionophore A23187 elicits normal aggregation and release reactions in the which is consistent with the observation presence of 400 fl arachidonste, that this ionophore does not activate platelets through the same membrane receptor system as physiological stimuli (16,17). What then is the significance of those observations? Arachidonate seems to be affecting receptorlnediated reactions by direct physical for arachidonate. interaction with the membrane, which would be a novel role Although other unsaturated fatty acids demonstrate similar effects (18), these are not likely to be physiologically significant since arachidonate fs the only fatty acid released from the platelet membrane upon stimulation Although an extracellular arachidonate concentration of 400 uM would (19). not be seen under physiological conditions, such levels may be reached locally via selective cellular compartmentalization and protein binding. Thus, the possible role of these observations in cell regulation must await further investigation.
REFERENCES 1.
SAMUELSSON, B. The role of prostaglandin endoperoxides and thromboxanes in human platelets. _In. --_ Prosta&andins in H_ematology. M.J. Silver, J.B. Smith and J.J. Rocsis. (E&s.), New York, 1977, Spectrum Publications, Inc. pp l-10.
2.
GORMAN, R.R. Modulation of thromboxane A2. Fed. Proc.
human platelet function 2~83-88, 1979.
by prostacyclin
and
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3. 'HAMBERG,M., SVENSSON, J., WAKABAYASHI, T. and SAMUELSSON, B. Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. Nat. Acad. Sci. USA z:345-349, 1974. 4.
SILVER, M.J., SMITH, J.B., INGERMAN, C. and KOCSIS, J.J. Arachidonic acid-induced human platelet aggregation and prostaglandin formation. Prostaglandins 4:863-875, 1973.
5.
VARGAFTIG, BB and ZIRINIS, P: Platelet aggregation induced by arachadonic acid is accompanied by release of potential inflammatory mediators distinct from PGE2 and PGF2= Nature New Biol. 244:114116, 1973.
6.
KLNLOUGH-RATHBONE, R.L., REIMERS, H.J., MUSTARD, J.F. and PACKHAM, M.A. Sodium arachidonate can induce platelet shape change and aggregation which are independent of the release reaction. Science 192:1011-1012, 1976.
7.
LINDER, B.L., CHERNOFF, A., KAPLAN, K.L. and GOODMAN, D.S. Release of platelet-derived growth factor from human platelets by arachidonic acid. Proc. Nat. Acad. Sci. USA 76:4107-4111, 1979.
8.
CLEMMONS, R.M., MEYERS, K.M. and KATZ, J. Formation of an aspirinsensitive, transferrable inhibitor of platelet aggregation from arachidonic acid by canine platelets. Fed. Proc. -391423, 1980, (abstract).
9.
KINLOUGH-BATHBORN, R.L., MUSTARD, J.F., PACKHAM, M.A., PERRY, D.W., REIMERS, H-J. and CAZENAVE, J-P. Properties of washed human platelets. Thromb. Haemostas. 2:291-308, 1977.
10. SMITH, J.B., INGERMAN, C.M. and SILVER, M.J. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J. Lab. Clin. Med. 88~167-172, 1976. 11. WROBLEWSKI, L. and LADUE, J.S. Lactic dehydrogenase activity in blood. Proc. Sot. Exp. Biol. Med. -90:210-213, 1955. 12. MOLNAR, J. and LO&?D, L. Studies on apyrases. Biophys. 2:353-363, 1961.
Arch. Biochem.
13. NIEWIAROWSKI, S., STEWART, G.J., NATH, N., SHA, A.T. and LIEBERMAN, G.E. ADP, thrombin and Bothrops atrox thrombinlike enzyme in plateletdependent fibrin retraction. Am. J. Physiol. -229:737-745, 1975. 14. DUTILH, C.E., HADDEMAN, E. and TENHOOR, F. Role of the arachidonate lipoxygenase pathway in blood platelet aggregation. In: Advances in Prostaglandin and Thromboxane Research, Vol. 6, Samuelsson, B., Ramwell, P.W., Paoletti, R., (Eds.), New York, 1980, Raven Press, pp. 101-105. 15. KINLOUGH-RATHBONE, R.L., CHANIL, A., PACKHAM, M.A. and MUSTARD, J.F. Conditions influencing platelet lysis. Lab. Invest. %:352-358, 1975.
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16.
WHITE, J.C., RAO G.&R. and GERRARD, J.M. Effects of the Ionophore A23187 on Blood Platelets. Am. J. Pathol. -77:1x-149, 1974.
17.
PACKHAM, M.A., CUCCIONE, M.A., GREENBERG, J.P., KINLOUGH-RATHBONE, R.L. MUSTARD, J.F. Release of [14C]-Serotonin during initial platelet changes induced by thrombin, collagen or A23187. -Blood 50: 915-926, 1977.
ia. MACINTYRE, D.E., HOOVER, R.L., KARNOVSKY, M.J., SALZMAN, E.W. Inhibition of platelet aggregation by unsaturated fatty acids. 'Circulation 62:111-191, 1980, (abstract). 19.
BILLS, T.K., SMITH, J.B., SILVER, M.J. Selective release of arachidonic acid from phospholipids of human platelets in response to thrombin. J. Clin. Invest. g:l-6, 1977.
CHARACTERIZATION OF THE PLATELET RESPONSE TO EXOGENOUS ARACHIDONIC ACID
Joseph C. Fratantoni and Betty J. Poindexter
Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland (Received 15.1.1981; in revised form 7.4.1981. Accepted by Editor J.S. Finlayson) ABSTRACT
When aggregation and release are the end points, washed human platelets treated with a range of concentrations of arachidonic acid display a bell-shaped dose-response curve. The peak is at approximately 200 PM, followed by a sharp decrease with minimum reaction at 400 uM. Prostaglandin synthesis, measured by malondialdehyde production, reaches peak activity at 400-500 uM and does not decrease. Platelet lysis, as manifested by lactate dehydrogenase release, increases only in response to arachidonate concentrations above 400 yM. When platelets are treated with 400 nM arachidonate for three minutes, subsequent reaction with thrombin, but not with A23187, is inhibited. This inhibitory activity is not sensitive to aspirin, indomethacin or ETYA and cannot be transferred in the supernatant fraction of the platelet suspension. The inhibitory activity generated from platelets reacted with 400 pM arachidonate is not a product of the cyclooxygenase or lipoxygenase systems.
INTRODUCTION The metabolic conversion of arachidonic acid to the prostaglandins and thromboxanes is causally associated with platelet aggregation and release reactions (1,Z). This pathway is physiologically significant since arachidonic acid is derived from membrane phospholipids via enzyme reactions which appear to be of fundamental importance in platelet activation. Several investigators have observed that addition of exogenous arachidonic acid to platelet preparations results in platelet reactions (3-6). Only recently, however, has the relationship between increasing concentration of arachidonic acid and platelet response been reported (7,8). When platelet release or aggregation is the measured reaction, addition of arachidonic acid (in the form of sodium arachidonate) yields a bell-shaped dose-response curve, that is, a biphasic response composed first of increasing, then of decreasing reaction intensity. Key Words:
Arachidonic Acid, Platelet Aggregation, Prostaglandins. 157
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In the course of studying the response of stored platelets to various stimuli, we have observed the biphasic relationship between aggregation or release, and arachidonate concentration. We have further noted that prostaglandin synthesis -_ per se, as indicated by malondialdehyde (MDA) production, does not demonstrate a biphasic response to progressive arachidonate levels. In this report, the following questions have been addressed regarding these observations: 1) Is the biphasic response the result of platelet lysis due to the detergent effect of sodium arachidonate? 2) Platelets exposed to higher concentrations of arachidonate display decreased reactions. Are such platelets similarly less reactive to other stimuli? 3) Is the altered platelet reactivity observed the result of metabolic products of arachidonate? MATERIALS AND _--METHODS Sample preparation Washed platelets (WP) were prepared by a modification of the method of Packham et al. (9). Blood from healthy donors who had ingested no known platelet-reactive medication for 10 days, except as noted, was collected into l/7 volume of citrate phosphate dextrose anticoagulant solution (CPD). Aspirin-treated platelets were obtained by administering 600 mg of aspirin to the donor two to four hours prior to venipuncture. Informed consent was obtained from each subject prior to the initiation of studies in accordance with the requirements of the Clinical Research Colrmittee. Platelet rich plasma (PRP) was separated at 200 x g for 15 min. [14C]serotonin (10'6w) and heparin (25 units/ml) were added and the mixture incubated for 30 minutes at 37 OC. The pH was adjusted to 6.3-6.5 with 0.25 M citric acid. The platelets were separated by centrifugation at 900 x g for 10 minutes and washed twice with Tyrode's buffer containing magnesium (1 mM), calcium (2 mM), apyrase (3-5 u/ml), and albumin (0.35 g/100 ml). Platelets were finally suspended in Tyrode's buffer, pH 7.4, containing albumin, magnesium, and calcium, as above, and apyraee (0.3-0.5 u/ml). WP were kept at 37 OC until used. All operations were done in polypropylene tubes (Falcon No. 2059 or 2063). Platelet aggregation Class cuvettes-and metal stir bars were siliconized. The aggregation response of 1 ml WP was observed in a Payton dual channel aggregation module (Payton Associates, Inc., Buffalo, NY). Aggregation was allowed to proceed for three minutes, at which time aliquots were removed and assayed as described below. Assay for released [14C]Serotonin --0.2 ml of reacted WP was added to 0.5 ml 0.13 M formaldehyde, with mixing. The mixtures were centrifuged at 2500 x g, for 10 minutes and the supernatant fluid counted in a Beckman liquid scintillation counter. Release due to factors other than arachidonic acid were accounted for with a saline control. A sample of labelled WP which had been lysed by mixing 1:lO with water was counted and used as the 100% value for calculation of percent release. Assay for MDA Production 0.7 ml of reactemP was taken from the cuvette, mixed into 0.7 ml 20%
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trichloroacetic acid in 0.6 M NC1 and assayed for MDA according to the method of Smith et al. (10). Tyrode's solution was used in place of platelet poor plasma for the controls. Difference spectra were performed on a Gary Model 219 spectrophotometer and A532 recorded. Lactate dehydrogenase Reagents and procedures from Sigma (LDH assay kit 340-UV) were used to perform a modification of the method of Wrobelewski et al. (11). The assay was standardized with Fisher Normal and Abnormal Clinical Chemistry Control Sera. Reagents Arachidonic acid, sodium salt was obtained from Nu-check, Elysian, MN, [14C]serotonin binoxalate from New England Nuclear Corp., and PentexB human albumin, fraction V, from Miles Laboratories. Apyrase was prepared from potatoes following the method of Molnar and Lorand (12), assayed according to Niewiarowski (13) and stored at -40 OC. Human thrombin, lot H-l, was provided by Dr. David Aronson, Bureau of Biologics. Eicosatetraynoic Acid (ETYA) was kindly provided by Dr. W. E. Scott, Hoffman-La Roche, Inc., Nutley, NJ).
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FIG. 1 Aggregation (0 -UJ), 114C]serotonin release (0 -0) and MDA production (A-A) response of washed platelets to sodium arachidonate. Values displayed are means of nine experiments using seven donors.
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RESULTS -Demonstration of the Biphasic Dose Response Curve Simultaneous measurement of aggregation, serotonin release, and MDA production in response to 0 to 400-cM arachidonic acid is shown in Figure 1. Al.1 donors tested demonstrated biphasic aggregation and release curves. Blood from a donor who had ingested 600 mg aspirin 12 hours prior to venlpuncture yielded platelets which were non-reactive to arachidonic acid at concentrations up to 400 PM. At arachidonate concentrations above 400 pM, serotonin was released at levels above 50% of total platelet radioactivity. Aggregation response to these concentrations varied from shape change alone to transmission readings above 60%. MDA production appeared to be maximal at 400 to 500 uM and did not change at higher concentrations. The release response to arachidonate levels >400 ufl was not decreased by prior ingestion of aspirin. The results of lactate dehydrogenase (LDH) asBay of the supernatant fractions of washed platelet suspensions are shown in Table 1. Platelet counts on suspensions of WP treated with 50-800 PM arachidonate showed no changes before and after When examined with phase microscopy, addition. it was noted that platelets treated with 600 PM arachidonate had lost their discoid shape and had become spherical. TABLE 1 LDH in Suuernatant Fraction of Platelet Susnensions St irked with Arachidonate for 3 Minute;. Final Concentration of Arachidonate CUM)
0
100 200 300 400 500 600 800
LDH (X of total in suspension + S.E.M)*
1.83 3.23 3.89 4.86 3.95 7.81 12.21 20.927
+ 7 + ? T T 7 -
1.2 1.8 0.9 1.3 1.2 1.0 1.7 1.5
* Total LDH in suspension determined by assay on sonicated samples which yielded a mean value of 645.5 international units/l012 platelets. Each value is the average of 4 or 5 assays.
These observations support the high concentrations of arachidonate specific lysis, and further suggest intact at these concentrations.
conclusions of Linder, et al. (7), that (in our system >400 PM) result In nonthat prostaglandin synthesis remains
Mechanism of the Biphasic Response Characzrization of the decreased aggregation response to high arachidonate levels was explored by reacting UP with 400 uM arachidonate then testing reactivity of the platelet suspension to thrombin. Figure shows the aggregation curves resulting from treatment of normal WP with
and 2A
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40 $1 or 400 pM arachidonate, followed after three minutes by addition of thrombin. The thrombin concentration required to achieve maximum aggregation after 400 nM arachidonate is 10 times that required after exposure to 40 nM arachidonate. A control (prior addition of buffer only) shows approximately the same reactivity to thrombin as the 40 pM arachidonate pretreatment sample.
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FIG. 2 Aggregation of washed platelets stirred with indicated concentrations of arachidonate (A) followed by thrombin (T). The interval between arachidonate and thrombin additions was three minutes. Results from one of six similar experiments. A: Normal platelets; B: Aspirin-treated platelets.
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In order to test the effects, on inhibition, of blocking the cyclooxygenase pathway inhibitors of this enzyme were added to the system. Figure 2B shows results of an experiment using WP derived from a donor who had ingested 600 mg of aspirin two hours prior to venipuncture. These platelets did not aggregate in response to 200 pM arachidonate. Other with similarly treated platelets, demonstrated that such cells experiments, or MDA responses to arachidonate at did not yield release, aggregation, concentrations from SO-400 pM. However , the inhibition of thrombin reactivity induced by pretreatment with 400 PM arachidonate is clearly present. The thrombin concentration required to stimulate maximum aggregation after 400 pM arachidonate pretreatment is approximately five times that required after 40 pM arachidonate pretreatment. The variability noted with other donors and the inherent problems of aggregation end points do not permit us to conclude from these data whether or not there is any difference between inhibition found with normal vs. aspirin-treated platelets. Addition of indomethacin in vitro, at a final concentration of 8 VU, similarly had no effect on the inhibition of thrombin reactions by e. 400 ~.IM arachidonat Arachidonate is also metabolized via the lipoxygenase system, which, in platelets, yields primarily a 12-hydroxylated derivative. This system can be inhibited by eicosatetraynoic acid (ETYA) at concentrations which do not fully inhibit the cyclooxygenase pathway (14). When 2.5 pM ETYA was added to WP, the amplitude of the aggregation response to 200 PM arachidonate was approximately one-half that of control WP. ETYA, at a final concentration of 2.5 nM was then added to a similar experimental system and the results are shown in Figure 3. The ETYA was added 1 minute prior to addition of arachidonate while a similar volume of solvent (DMSO) was added as a control. system did not differ from the control in any The ETYA treated way. Finally, the calcium ionophore A23187 was used as the test stimulant in place of thrombin. Concentrations from 0.1 to 5.0 PM A23187 gave progressively increasing aggregation and release responses which were not affected by prior addition of 400 pM arachidonate. In order to test for a soluble, extracellular inhibitor, WP from normal and aspirin-treated donors were reacted with either buffer, 40 PM arachidonate or 400 uM arachidonate for three minutes, then centrifuged at 900 x g, for 10 minutes, at 22 OC. The supernatants were mixed with an equal volume of WP derived from an aspirin-treated donor. Aspirin-inhibited platelets were used as the test system to avoid stimulatory effect of arachidonate (in the supernatant) on aggregation. The platelets were tested within 60 minutes of resuspension. Eleven such experiments were performed using platelets from nine subjects. The supernatant fractions from cells treated with either buffer or 40 IJM arachidonate, when mixed with test cells, reacted similarly with thrombin, showing aggregation >80% of maximum at threshold concentrations of 0.05 to 0.1 units of thrombiniml. The addition to test cells of supernatant fractions from platelets treated with 400 pM arachidonate gave more variable results. In two experiments, the buffer control threshold concentration was 0.06 units/ml, while the threshold with the 400 PM arachidonate supernatant was 0.1 unit/ml. In the remainder of the experiments, no consistent pattern of inhibition by supernatant from cells treated with 400 pM arachidonate was seen, even when these cell suspensions contained lOa platelets/ml. In order to test for the presence of activators in the supernatant fractions, 400 pM arachidonate was stirred for three minutes at 37 Oc with either normal platelets, aspirin-treated platelets or Tyrode’s buffer. An aliquot of supernatant from each mixture was added to an equal volume of
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washed normal platelets and aggregation and release measured. The solutions which had been in contact with platelets for three minutes, when added to test cells, gave 0 to 4% aggregation and 0 to 6.3% release. The Tprode's buffer control which contained a final arachidonate concentration of 200 uM after mixing with the test platelets, gave 57% aggregation and 48% release.
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FIG. 3 Aggregation of washed, aspirin-treated platelets with indicated concentration of arachidonate (A) and Thrombin (T). ETYA (E) was added as 10 microliters of a 0.25 mM solution in DMSO. *lO microliters of DMSO added to sample 3 at "E".
DISCUSSION -Addition of arachidonate to washed hrrmanplatelets results in measurable aggregation, release of intracellular serotonin and formation of MDA. In these studies we have observed that 1) the arachidonate dose-response curve is bell-shaped if aggregation or release are measured, but not when MDA production is the end point; 2) exposure of platelets to 400 pM arachidonate results in inhibition of subsequent reactivity to thrombin but not to the calcium ionophore A23187; 3) the inhibitory activity is not affected by prior treatment with aspirin, indomethacin or ETYA; 4) the inhibitory activity could not be demonstrated in the plateletarachidonate supernatant; and 5) the effects seen as a result of 400 PM arachidonate are not due to platelet lysis. The potency of arachidonate in this system is a function of the protein concentration in the reaction medium. In the final suspension buffer employed, containing 3.5 g of albumin per liter, the nadir of platelet reactivity at 400 uM is consistent with previous reports, using either low albumin buffers or plasma (7). In our hands, this inhibition is not affected by aspirin or indomethacin. Clemmons, et al. (8) report that their inhibitor is aspirin sensitive, but their system is quite different (dog platelets, PRP, serotonin stimulation) and the limited data available do not permit further explanation.
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The determination of LDH in the supernatant fraction of WP treated with arachidonate clearly demonstrates that cell lysis is not a factor with arachidonate concentrations <400 w. Kinlough-Rathbone et al. (15) have shown that measurable lysis accompanies the release reaction stimulated by Our data similarly show the lowest LDH levels in the thrambin or collagen. buffer control sample, but also show no differences in LDH from samples treated with all concentrations of arachidonate 5400 UM. Aa the concentrations of arachidonate are raised above 400 $f, increasing lysis The aspirin-insensitive phenomonena occurs manifested by higher LDH levels. observed with the aggregometer and [ 14C]serotonin procedures are thus most readily explained by cell lysis. Experiments which utilized the supernatant fractions from platelet suspensions treated with 400 IJMarachidonate did not clearly demonstrate a At first these aggregation results were not as soluble inhibitor. reproducible as other studies. Very careful attention to sample preparation (uniform platelet count, samples to be compared run simultaneously, identical mixing times for test and control samples) eliminated some of this variation. We believe additional problems result from constituents released from the As can be seen in Fig. 1, platelet upon treatment with 400 I.IMarachidonate. release is inhibited at that level of arachidonate but is not zero, and it varies with time and with the subject. Such factors may account for the observations by others (7,B) of a soluble inhibitor. Agents known to block arachidonate metabolism via the cyclooxygenase pathway (aspirin, indomethacin) or the lipoxygenase pathway (ETYA) did not interfere with the inhibition observed with 400 $i arachidonate. MDA production is not inhibited, and is evidence that arachldonate is Therefore, the metabolized to products of the cyclooxygenase pathway. platelet must be prevented from undergoing thrombin-stimulated aggregation and release reactions by its interaction with arachidonate itself. The ionophore A23187 elicits normal aggregation and release reactions in the which is consistent with the observation presence of 400 fl arachidonste, that this ionophore does not activate platelets through the same membrane receptor system as physiological stimuli (16,17). What then is the significance of those observations? Arachidonate seems to be affecting receptorlnediated reactions by direct physical for arachidonate. interaction with the membrane, which would be a novel role Although other unsaturated fatty acids demonstrate similar effects (18), these are not likely to be physiologically significant since arachidonate fs the only fatty acid released from the platelet membrane upon stimulation Although an extracellular arachidonate concentration of 400 uM would (19). not be seen under physiological conditions, such levels may be reached locally via selective cellular compartmentalization and protein binding. Thus, the possible role of these observations in cell regulation must await further investigation.
REFERENCES 1.
SAMUELSSON, B. The role of prostaglandin endoperoxides and thromboxanes in human platelets. _In. --_ Prosta&andins in H_ematology. M.J. Silver, J.B. Smith and J.J. Rocsis. (E&s.), New York, 1977, Spectrum Publications, Inc. pp l-10.
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GORMAN, R.R. Modulation of thromboxane A2. Fed. Proc.
human platelet function 2~83-88, 1979.
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and
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3. 'HAMBERG,M., SVENSSON, J., WAKABAYASHI, T. and SAMUELSSON, B. Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. Nat. Acad. Sci. USA z:345-349, 1974. 4.
SILVER, M.J., SMITH, J.B., INGERMAN, C. and KOCSIS, J.J. Arachidonic acid-induced human platelet aggregation and prostaglandin formation. Prostaglandins 4:863-875, 1973.
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VARGAFTIG, BB and ZIRINIS, P: Platelet aggregation induced by arachadonic acid is accompanied by release of potential inflammatory mediators distinct from PGE2 and PGF2= Nature New Biol. 244:114116, 1973.
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KLNLOUGH-RATHBONE, R.L., REIMERS, H.J., MUSTARD, J.F. and PACKHAM, M.A. Sodium arachidonate can induce platelet shape change and aggregation which are independent of the release reaction. Science 192:1011-1012, 1976.
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LINDER, B.L., CHERNOFF, A., KAPLAN, K.L. and GOODMAN, D.S. Release of platelet-derived growth factor from human platelets by arachidonic acid. Proc. Nat. Acad. Sci. USA 76:4107-4111, 1979.
8.
CLEMMONS, R.M., MEYERS, K.M. and KATZ, J. Formation of an aspirinsensitive, transferrable inhibitor of platelet aggregation from arachidonic acid by canine platelets. Fed. Proc. -391423, 1980, (abstract).
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KINLOUGH-BATHBORN, R.L., MUSTARD, J.F., PACKHAM, M.A., PERRY, D.W., REIMERS, H-J. and CAZENAVE, J-P. Properties of washed human platelets. Thromb. Haemostas. 2:291-308, 1977.
10. SMITH, J.B., INGERMAN, C.M. and SILVER, M.J. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J. Lab. Clin. Med. 88~167-172, 1976. 11. WROBLEWSKI, L. and LADUE, J.S. Lactic dehydrogenase activity in blood. Proc. Sot. Exp. Biol. Med. -90:210-213, 1955. 12. MOLNAR, J. and LO&?D, L. Studies on apyrases. Biophys. 2:353-363, 1961.
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13. NIEWIAROWSKI, S., STEWART, G.J., NATH, N., SHA, A.T. and LIEBERMAN, G.E. ADP, thrombin and Bothrops atrox thrombinlike enzyme in plateletdependent fibrin retraction. Am. J. Physiol. -229:737-745, 1975. 14. DUTILH, C.E., HADDEMAN, E. and TENHOOR, F. Role of the arachidonate lipoxygenase pathway in blood platelet aggregation. In: Advances in Prostaglandin and Thromboxane Research, Vol. 6, Samuelsson, B., Ramwell, P.W., Paoletti, R., (Eds.), New York, 1980, Raven Press, pp. 101-105. 15. KINLOUGH-RATHBONE, R.L., CHANIL, A., PACKHAM, M.A. and MUSTARD, J.F. Conditions influencing platelet lysis. Lab. Invest. %:352-358, 1975.
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WHITE, J.C., RAO G.&R. and GERRARD, J.M. Effects of the Ionophore A23187 on Blood Platelets. Am. J. Pathol. -77:1x-149, 1974.
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PACKHAM, M.A., CUCCIONE, M.A., GREENBERG, J.P., KINLOUGH-RATHBONE, R.L. MUSTARD, J.F. Release of [14C]-Serotonin during initial platelet changes induced by thrombin, collagen or A23187. -Blood 50: 915-926, 1977.
ia. MACINTYRE, D.E., HOOVER, R.L., KARNOVSKY, M.J., SALZMAN, E.W. Inhibition of platelet aggregation by unsaturated fatty acids. 'Circulation 62:111-191, 1980, (abstract). 19.
BILLS, T.K., SMITH, J.B., SILVER, M.J. Selective release of arachidonic acid from phospholipids of human platelets in response to thrombin. J. Clin. Invest. g:l-6, 1977.