role of arachidonic acid metabolism in human platelet activation and irreversible aggregation

American Journal of Hematology 19:339-347 (1985)

Role of Arachidonic Acid Metabolism in Human Platelet Activation and Irreversible Aggregation Gundu H.R. Rao and James G. White

Departments of Pediatrics (J.G. U) and Laboratory Medicine and Pathology (G. H. R. R.), University of Minnesota Health Sciences Center, Minneapolis

Previous studies from our laboratory have demonstrated that the aggregation response of platelets inhibited by agents blocking cyclooxygenase activity could be restored to a normal state of sensitivity by prior stimulation of a-adrenergic receptors. Since cyclooxygenase activity and thromboxane synthesis are not absolutely required for irreversible platelet aggregation, it is important to define precisely what role this pathway serves in platelet physiology. The present study has evaluated the influence of agents that selectively block arachidonic acid conversion at different steps of synthesis. Inhibition of peroxidase, cyclooxygenase, lipoxygenase, and thromboxane synthetase blocked the second wave response of platelets to several agonists, but did not cause dissociation of aggregates preformed by prior exposure to arachidonate (AA) or adenosine diphosphate. Phospholipase (A*/C) inhibitors, similar to prostaglandin inhibitors, blocked the second wave response of platelets to the action of agonists and, in addition, caused dissociation of aggregates induced by aggregating agents. Results of our study demonstrate that when single agonists are tested at threshold concentrations, products of arachidonate metabolism may play a role in the activation process. However, continued generation of these metabolites does not appear to be essential for the maintenance of irreversible aggregation. When a combination of agents or high concentration of physiological agonists are used, both activation and irreversible aggregation can be secured independent of prostaglandin synthesis or the release reaction.

Key words: prostaglandins, platelet activation, irreversible aggregation

INTRODUCTION

Studies from several laboratories have proposed a definitive role for arachidonic acid and its metabolites in inducing shape change, secretion of granule contents, and aggregation of blood platelets [ 1-91. Platelets mobilize arachidonic acid in response to the action of a majority of agonists, and the substrate released from platelet membrane phospholipids is rapidly converted to transient intermediates (cyclic endoperoxides PGG2 and PGH2) by cyclooxygenase and then to thromboxane A2 by thromboxane synthetase [ 101. The major biologically active metabolite of arachidonic acid metabolism in platelets is thromboxane A2 [ 111.

Platelets from most dogs do not develop irreversible aggregation when stirred with arachidonate [ 121. However, the nonresponding platelets generate as much

Received for publication September 27,1984; accepted January 31, 1985.

Address reprint requests to Gundu H.R. Rao, Ph.D., Department of Laboratory Medicine and Pathology, Box 198 Mayo Memorial Building, 420 Delaware Street S.E., Minneapolis, MN 55455

0 1985 Alan R. Liss, Inc.

340 Rao and White

thromboxane A2 as the platelets which aggregate in response to stimulation by arachidonate [ 131. Platelets from patients who are deficient in cyclooxygenase activity do not aggregate irreversibly in response to arachidonate [ 141. However, in patients and canines, exposure of arachidonate refractory platelets to epinephrine restored the response of platelets to the action of arachidonate [14,15]. Similarly, the platelets exposed to aspirin also could be made to respond to the action of agonists by exposing drug-suppressed platelets to the action of epinephrine and then challenging with other agonists [ 16,171. Although in these different situations platelets are refractory to the action of arachidonate, in none of these situations does severe bleeding result under normal conditions.

To clarify the role of arachidonic acid metabolites in irreversible aggregation of platelets, we have evaluated the effect of various inhibitors of arachidonic acid cascade on platelet function. Results of our studies show that arachidonic acid and its metabolites play a role in inducing shape change and activation of platelets. However, continued generation of these metabolites does not appear to be essential for the maintenance of irreversible aggregation.

MATERIALS AND METHODS Materials

Arachidonic acid as the sodium salt was obtained from Nu Chek Prep, (Elysian, MN) and made up in 0.1 M Tris buffer at pH 7.6. Prostacyclin as a sodium salt was from The Upjohn Company (Kalamazoo, MI). Thromboxane/endoperoxide receptor antagonist 13-azaprostanoic acid was a gift from Professor Guy C. LeBreton (Depart- ment of Pharmacology, University of Illinois, Chicago, IL). Unless otherwise stated, all other chemicals were from Sigma Chemical Company (St. Louis, MO).

Methods

Blood for these studies was obtained from healthy adult volunteers after in- formed consent. None of these subjects had taken any drugs within 10 days prior to venesection. Blood drawn from an anticubital vein into plastic syringes was mixed immediately with sodium citrate-citric acid-dextrose (citrate 100 mM, citric acid 7mM, dextrose 140 mM, CCD, pH 6.5) in a ratio of 9 parts blood to 1 part CCD. Platelet-rich plasma (PRP) was separated by centrifugation of whole blood at room temperature for 20 min at 100 x g. Platelet aggregation studies were carried out on a Payton dual-channel aggregometer present with PRP and platelet-poor plasma [ 18,191. Where needed, appropriate concentrations of inhibitors were added before or after the agonists to cuvettes containing PRP, and the response was followed. All aggregation studies were repeated at least three times and often more than five times. The figures represent a typical response of aggregates to the action of inhibitory drugs following platelet activation.

RESULTS Effect of Inhibitors on Platelet Response to Agonists

Normal control platelets aggregated irreversibly in response to epinephrine (5 pM), adenosine diphosphate (ADP, 3 pM), and arachidonate (0.45 mM). Inhibitors such as prostacyclin (10 nM), phenacyl bromide, mepacrine, chlorpromazine, stela-

Role of Eicosanoids in Platelet Function 341

zine (0.2mM), eicosatetraynoic acid (ETYA, 0.2mM), aminotriazole (1 mM), aspirin (100 pM), indomethacin (10 pM), irnidazole (100 pglml), and 13-azaprostanoic acid (100 pM) effectively blocked arachidonic acid induced platelet aggregation.

Effect of Phosphoiipase inhibitors on Arachidonate-Induced Aggregates

In our earlier study we demonstrated that the dissociating effect of antiplatelet drugs on preformed platelet aggregates can be studied on an aggregometer by the addition of drugs after the response of platelets to agonists has reached a plateau [20]. Using this system, it was found that phospholipase inhibitors, including phenacyl bromide, mepacrine, and chlorpromazine at concentrations of 0.5 mM, caused dissociation of arachidonate-induced disaggregates (Fig. 1). Agents that stimulated adenylate cyclase, such as prostacyclin (70 nM), did not dissociate aggregates induced by arachidonate (Fig. 2).

Inhibitors of Arachidonic Acid Converting Enzymes and Platelet Dissociation

Arachidonic acid is metabolized in platelets by the lipoxygenase and cyclooxygenase pathways. Conversion of substrate by these pathways is blocked by eicosatetraynoic acid (1 mM). The competitive inhibitor of the substrate arachidonic acid dissociated aggregates stimulated by ADP but failed to dissipate arachidonate induced aggregates (Fig. 3). Previous studies from our laboratory showed a critical role for peroxidase in arachidonic acid metabolism. However, the peroxidase inhibitor, ami-

I Phenocyl bromide

A T * change in light transmission

Fig. 1. Influence of a phospholipase inhibitor (phenacyl bromide), membrane active drugs (mepacrine, chlorpromazine), and a calmodulin antagonist (stelazine) on disaggregation of arachidonate induced aggregates. The drugs were added at the same concentrations (0.5 mh4) to separate samples of c-PRP just after the recorded response of the sample to arachidonate (0.5mM) had reached maximum amplitude on the aggregometer. Since all four drugs yielded an identical response, only one tracing is shown. The several agents all caused rapid dissociation of aggregates induced by arachidonate.

342 Rao and White

AA0.45mM ' f \

*AT- change in light transmission

Fig. 2. Influence of an adenylate cyclase stimulator (prostacyclin, PG12) on preformed aggregates induced by adenosine diphosphate and arachidonate. Prostacyclin was added to platelets in plasma just after the response of platelets to agonists had reached a maximum. The drug caused deaggregation of ADP aggregates but had no such influence on arachidonate aggregates.

AT *

AT*-change in light transmission

Fig. 3. Influence of an inhibitor of lipoxygenase and cyclooxygenase enzymes on the aggregates induced by ADP and arachidonate. Eicosatetraynoic acid (ETYA) was added to samples of platelets in plasma soon after the response of cells to agonists had reached a maximum. ETYA caused rapid deaggregation of ADP aggregates but was unable to dissociate arachidonate aggregates.

notriazole (1 mM), did not cause dissociation of arachidonate induced aggregates (Fig. 4).

Cyclooxygenase and Thromboxane Synthetase Inhibitors and Platelet Aggregate Dispersal

Cyclooxygenase inhibitors, aspirin ( 100 pM) and indomethacin (10 pM) , were unable to dissociate arachidonate aggregates (Fig. 4). Similarly, thromboxane synthe-


AT * Amlnotrlb.zo/e I x ?O-jM

- 1 minute 0.5mM AA .f


Fig. 4. Influence of cyclooxygenase inhibitors (aspirin, indomethacin) and a peroxidase inhibitor (aminotriazole) on aggregates induced by ADP and arachidonate. These drugs were individually tested with separate samples of platelets in plasma. The figure represents a typical response of aggregates to the action of inhibitory drugs following platelet activation. None of the drugs at the concentration tested caused deaggregation of performed aggregates.

AT * lOOpq/m/ fmi dm0 fe IO/M 9,I I - A 20 - I 50pM / ~ - A Z O ~ ~ O S ~ O ~ O I C ACld 7OOpM OKY i58t OKY046 -

1 minute

O . 5 n M A A 4


Fig. 5. Influence of thromboxane synthetase inhibitors (9,11-Azo-l, imidazole, OKY 046, OKY 1581) and a thromboxane receptor antagonist (13-azaprostanoic acid) on aggregates induced by ADP and arachidonate. These drugs at different concentrations were added to separate samples of platelets in plasma soon after the response of platelets had reached a maximum. None of the compounds tested could induce deaggregation of preformed platelet aggregates. Tracings of typical responses obtained with these drugs are shown.

tase inhibitors, imidazole (100 pg/ml) and OKY compounds (OKY 046, OKY 1581; 100 pM), did not induce dispersal of arachidonate induced aggregates (Fig. 5) .

Effect of Thromboxane Receptor Antagonism on Arachidonate Aggregates LeBreton and Venton have demonstrated that 13-azaprostanoic acid (13-APA) at

100 p M concentration effectively reversed aggregates induced by epinephrine, ADP

344 RaoandWhite

and arachidonate [22]. In the present study, 13-APA antagonized the effects of arachidonate when added in the first few seconds after activation but had no disaggre- gating effect when tested at 1 min or later following platelet stimulation.

DISCUSSION

Current concepts of factors involved in irreversible aggregation of blood platelets envisage three independent mechanisms: secretion of adenosine diphosphate, synthesis of thromboxane, and formation of platelet-activating factor [23,24]. More recent studies from our laboratory demonstrated another intrinsic mechanism, re- ferred to as membrane modulation, capable of securing irreversible platelet aggregation independent of ADP, thromboxane, or platelet-activating factor [25,26].

Thromboxane A*, the major metabolite of arachidonic acid conversion via the cyclooxygenase pathway, has similar properties as its precursors , PGGz and PGH2 [4-61. These compounds induce platelet release reaction and cause irreversible aggregation [l-31. Synthesis of these compounds is considered essential for the normal function of platelets as deficiency in cyclooxygenase activity has been considered responsible for defective platelet release and prolonged bleeding [ 14,27-30]. How- ever, in an earlier study from our laboratory, we reported a patient with cyclooxygenase deficiency with a normal bleeding time [ 151. In addition, several studies from our laboratory have demonstrated that the sensitivity of refractory platelets to the action of agonists could be restored by epinephrine-induced membrane modulation [ 15-18, 31-34]. The present study, to a great extent, was stimulated by the discovery of membrane modulation, which can secure irreversible aggregation in the absence of release reaction and thromboxane synthesis. In the present investigation, we have sought to determine the specific role of the active intermediates of arachidonic acid metabolism in sustaining irreversible aggregation.

Results of our study demonstrate an important role for the active intermediates of arachidonic acid metabolism in platelet activation and the release reaction. Inhibi- tion of arachidonate release or conversion of the substrate to the active intermediates, such as endoperoxides/thromboxane, prevented platelet release reaction and irreversible aggregation. These inhibitors have been shown to block the second-wave response of threshold concentrations of epinephrine, thrombin, ADP, and platelet- activating factor effectively, suggesting their dependence upon active intermediates of arachidonate metabolism for inducing release reaction [26,36]. We also have shown in our earlier studies that epinephrine could restore the sensitivity of cyclooxygenase deficient platelets to the action of arachidonate and other agonists [15]. Our studies, as well as those of others, have shown that agonists at higher concentrations can also overcome the dysfunction caused by inhibition of arachidonate metabolism [ 16,17,25,35]. Therefore, it is reasonable to speculate that in normal physiological conditions where threshold concentrations of agonists activate platelets, arachidonate metabolites play an important role in potentiating the action of other agonists. However, when high concentrations of an agonist, such as thrombin, is used, or membrane modulation is initiated, activation and irreversible aggregation of platelets can be secured independent of prostaglandin synthesis and the release reaction [15- 18,20,31,32,34].

Studies using phospholipase inhibitors, such as phenacyl bromide, mepacrine, and chlorpromazine, showed their effectiveness in causing deaggregation of platelet


clumps caused by arachidonate. On the other hand, adenylate cyclase stimulators, such as prostacyclin, prostaglandin (PG) El and PGD2 were less effective in causing deaggregation of arachidonate aggregates. Both classes of compounds seem to share a common mechanism of action [39,40]. They have been shown to inhibit the release of arachidonic acid from membrane phospholipids by preventing the activation of phospholipases. However, this known mechanism of action fails to provide adequate explanation as to how these compounds induce deaggregation of platelet clumps because, when these drugs are added to platelets, the activation of platelets and the liberation of arachidonic acid has already taken place. Therefore, their deaggregating effect of platelet clumps may be on a postactivation step related to some specific alteration they induce in the membrane. Rao et al [ 181 in an earlier study demonstrated that prostacyclin could induce specific refractoriness to the action of arachidonate, and this effect of prostacyclin is not mediated by significant elevation of cyclic AMP. Similarly, other adenylate cyclase stimulators, as well as phospholipase inhibitors, may induce alterations in the membrane and thereby interfere with a critical step, such as fibrinogen binding, membrane calcium availability, or calcium flux [34].

Inhibitors of peroxidase, cyclooxygenase, and thromboxane synthetase did not exert any dissociating effect upon arachidonate induced platelet aggregates. These results suggest that once the metabolites of arachidonate are generated, to activate platelets, further synthesis is either not essential or does not take place. A prostaglandin endoperoxide/thromboxane receptor antagonist had a limited effect on platelet aggregates. Its ability to dissociate aggregates was limited to the first 30 sec following platelet activation. Similar results have been observed by other workers, suggesting that thromboxane receptor antagonism leading to the dispersal of aggregates can be achieved for only a few seconds following activation [22]. Receptor occupancy may be essential for initiating activation of platelets. Once the receptor is occupied by thromboxane, addition of an antagonist, such as 13-APA, may not effectively displace the agonist.

In conclusion, results of our study demonstrate an important role for the active intermediates of arachidonic acid metabolism, Inhibition of arachidonic acid release or the conversion of the substrate into endoperoxide/thromboxane, prevents platelet release reaction as well as irreversible aggregation. Although these metabolites play a critical role in causing activation of platelets, their continued generation may not be essential for sustaining irreversible aggregation. Inhibition of any of the enzymes involved in arachidonic acid metabolism, after the initiation of platelet activation, has very little influence on the preformed aggregates. Agents that deaggregate arachidonate aggregates may therefore interfere with some critical step (after platelet activation) essential for sustaining irreversible aggregation.

ACKNOWLEDGMENT

The authors would like to thank Ms. Lori Anderson for her technical assistance. This work was supported by USPHS grants H2-11880, HL-16833, CA-21737,

GM-22167, AM-17697, HL-30217, and a grant to Dr. Rao from the American Heart Association Minnesota affiliate.

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role of arachidonic acid metabolism in human platelet activation and irreversible aggregation

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