Indomethacin-treated Platelets Research Articles

Purification, Cloning, Expression, and Mechanism of Action of a Novel Platelet Aggregation Inhibitor from the Salivary Gland of the Blood-sucking Bug, Rhodnius prolixus

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.

Selective inhibition of collagen-induced arachidonic acid liberation by 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1, 4-diazepine hydrochloride (ML-7), a myosin light chain kinase inhibitor, in washed rabbit platelets

Effects of myosin light chain (MLC) kinase inhibitor ML-7 [1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1, 4-diazepine hydrochloride] and protein kinase C inhibitor H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride] on collagen-induced platelet activation were investigated in washed rabbit platelets. Upon stimulation with collagen (1μg/mL), H-7 decreased protein kinase C-mediated pleckstrin phosphorylation, but had no inhibitory effect on thromboxane (TX) A 2 formation or platelet aggregation. In contrast, ML-7 produced a concentration-dependent inhibition of the collagen-induced platelet aggregation and TXA 2 formation by preventing arachidonic acid (AA) liberation from membrane phospholipids. However, ML-7 had little effect on AA liberation induced by thrombin, Ca 2+ ionophore A-23187 or melittin, suggesting that ML-7 may affect the signal transduction pathway specific for collagen-induced AA liberation, without direct inhibition of phospholipase A 2 activity. In indomethacin-treated platelets, collagen caused MLC phosphorylation and AA liberation in the absence of a significant increase in intracellular Ca 2+ concentration ([Ca 2+] i) or protein tyrosine phosphorylation. ML-7 inhibited both MLC phosphorylation and AA liberation induced by collagen in indomethacin-treated platelets. These results demonstrate that MLC phosphorylation and AA liberation are early events detectable in collagen-stimulated platelets, and suggest that ML-7 inhibits these early steps of collagen-induced signal transduction pathway in rabbit platelets.

CGRP enhances induction of NO synthase in vascular smooth muscle cells via a cAMP-dependent mechanism.

Experiments were designed to examine whether calcitonin gene-related peptide (CGRP), a potent adenosine 3',5'-cyclic monophosphate (cAMP)-dependent vasodilator, affects the production of NO evoked by interleukin-1 beta) (IL-1 beta) in cultured rat aortic smooth muscle cells (SMC). CGRP, in a concentration-dependent manner, enhanced the release of nitrite (a stable oxidation product of NO) and the formation of L-citrulline from L-arginine caused by IL-1 beta. Two cAMP-dependent vasodilators, forskolin and isoproterenol, and the activator of the cAMP-dependent protein kinase, Sp-cAMPS, also enhanced the release of nitrite and the formation of L-citrulline evoked by IL-1 beta. The enhancing effect of isoproterenol required the presence of the vasodilator during the induction of NO synthase (NOS). IL-1 beta-treated vascular SMC inhibited the aggregation of indomethacin-treated platelets. Inhibition of platelet aggregation was more marked with SMC exposed to a combination of IL-1 beta and either CGRP or isoproterenol than with cells exposed to IL-1 beta alone. This inhibition was prevented by methylene blue and oxyhemoglobin. IL-1 beta induced the expression of inducible NOS mRNA in vascular SMC, which was enhanced by coincubation of IL-1 beta with either CGRP, isoproterenol, or forskolin. These observations indicate that CGRP via a cAMP-dependent mechanism potentiates the IL-1-beta-induced production of NO by enhancing the expression of inducible NOS. Therefore CGRP may contribute to the substantial production of NO in the vasculature during septic shock, which accounts, at least in part, for the collapse of the vascular system.

Insulin-like growth factor I inhibits induction of nitric oxide synthase in vascular smooth muscle cells.

Experiments were designed to examine whether or not insulin-like growth factor I (IGF-I), which is produced by vascular cells in response to injury, affects the production of nitric oxide evoked by the inducible nitric oxide synthase in cultures of smooth muscle cells from the rat aorta. Nitric oxide production was assessed indirectly by the measurement of nitrite accumulation and nitric oxide synthase activity by determining the formation of L-citrulline from L-arginine. Nitric oxide synthase was induced in vascular smooth muscle cells that had been exposed to interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha). IGF-I inhibited, in a concentration-dependent manner, the production of nitrite and L-citrulline evoked by IL-1 beta or TNF-alpha. The inhibition caused by IGF-I required the presence of the growth factor during the induction of nitric oxide synthase. Two IGF-I-related proteins, IGF-II and insulin, also inhibited, but to a smaller extent, the release of nitrite and the formation of L-citrulline stimulated by IL-1 beta. Under bioassay conditions, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed rings of rat aorta without endothelium that had been contracted with phenylephrine; these relaxations were reversed by nitro-L-arginine. Addition of IL-1 beta-treated vascular smooth muscle cells to indomethacin-treated platelets inhibited their aggregation to thrombin; methylene blue prevented this inhibition. Control smooth muscle cells or cells exposed to IGF-I alone did not have such effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin inhibits induction of nitric oxide synthase in vascular smooth muscle cells

Experiments were designed to examine whether thrombin affects the production of nitric oxide-like factor(s) evoked by interleukin-1 beta (IL-1 beta) in cultured smooth muscle cells from the rat aorta. IL-1 beta stimulated the release of nitrite (a stable oxidation product of nitric oxide) from cultured smooth muscle cells. Thrombin inhibited in a concentration-dependent manner the release of nitrite caused by IL-1 beta. The inhibition was prevented by hirudin (a thrombin inhibitor) and required the presence of thrombin before or during the induction period. Under bioassay conditions, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector rat aortic rings without endothelium. The addition of IL-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated platelets inhibited their aggregation. Control untreated smooth muscle cells or cells treated with thrombin alone did not have such effects. The treatment of smooth muscle cells with IL-1 beta in combination with thrombin blunted both the relaxing activities of the perfusates under bioassay conditions and the inhibition of platelet aggregation. These observations indicate that thrombin inhibits the production of nitric oxide-like factor(s) evoked by the inducible nitric oxide synthase in cultured smooth muscle cells from rat aorta.

Plasmin potentiates induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells.

Experiments were performed to examine the effect of the major fibrinolytic protease, plasmin, on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured human and rat aortic smooth muscle cells. Incubation of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite and nitrate in the culture media. Plasmin, either added exogenously or generated by the reaction of tissue plasminogen activator with plasminogen, potentiated the IL-1 beta-mediated release of nitrite and nitrate from smooth muscle cells in a concentration-dependent manner, without affecting the production of nitrite and nitrate from cells untreated with IL-1 beta. This potentiating effect was abolished when plasmin was incubated with the protease inhibitor, alpha 2-antiplasmin. The perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium, and the addition of IL-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated platelets inhibited their aggregation. Untreated smooth muscle cells or cells treated with plasmin alone did not have such effects. However, the simultaneous treatment of smooth muscle cells with IL-1 beta and plasmin markedly enhanced both the relaxing activities of the perfusates and the inhibition of platelet aggregation. Treatment of smooth muscle cells with NG-nitro-L-arginine inhibited the cytokine-mediated effects as well as the potentiating effect of plasmin. These results demonstrate that the plasmin can enhance the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells.

Eicosapentaenoic acid potentiates the production of nitric oxide evoked by interleukin-1 beta in cultured vascular smooth muscle cells.

Experiments were designed to determine whether the omega 3-unsaturated fatty acid eicosapentaenoic acid affects the production of nitric oxide evoked by interleukin-1 beta in cultured vascular smooth muscle cells. Incubation of cultured rat or human aortic smooth muscle cells with interleukin-1 beta evoked a time- and concentration-dependent release of nitrite, an oxidation product of nitric oxide. The exposure of cells to interleukin-1 beta in combination with eicosapentaenoic acid caused a significantly larger production of nitrite than that evoked by the cytokine alone. The potentiation by eicosapentaenoic acid was concentration-dependent. The production of nitrite evoked by equieffective concentrations of interleukin-1 beta in the presence and absence of eicosapentaenoic acid were inhibited to a similar extent by nitro L-arginine (an inhibitor of nitric oxide synthase), transforming growth factor beta 1, platelet-derived growth factorAB and thrombin. The addition of interleukin-1 beta-activated smooth muscle cells to suspensions of washed and indomethacin-treated platelets inhibited the aggregation caused by thrombin. The inhibitory effect was enhanced when the smooth muscle cells were exposed to the cytokine in the presence of eicosapentaenoic acid prior to the experiment. Smooth muscle cells exposed to interleukin-1 beta and eicosapentaenoic acid did not affect platelet aggregation in the presence of oxyhemoglobin or methylene blue. Untreated cells or cells exposed to the fatty acid alone did not have such effects. These observations suggest that eicosapentaenoic acid potentiates the production of nitric oxide evoked by interleukin-1 beta in vascular smooth muscle.

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619.

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.

Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5′-[β-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction

The effect of guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), reported to be an antagonist of GTP at the G-protein-binding site, on human platelet activation was examined. GDP[beta S] (0.3-3 mM) had significant inhibitory effects on platelet aggregation and 5-hydroxytryptamine (5HT) secretion induced by thrombin, collagen, the thromboxane mimetic U46619 and 1,2-dioctanoylglycerol (diC8) in intact platelets, as well as in saponin-permeabilized platelets. Similar inhibitory effects in intact platelets were also observed with ATP (over similar concentration ranges) and GDP and GTP (at 2- and 10-fold higher concentrations respectively). All four nucleotides also inhibited ADP-induced platelet aggregation in indomethacin-treated platelets under conditions where no 5HT secretion occurred. Inhibition of thrombin-induced aggregation and secretion by GDP[beta S] and ATP in intact platelets was accompanied by a reduction in the thrombin-induced rise in intracellular Ca2+ levels and 45 kDa-protein phosphorylation. The results suggest that at least some of the effects of GDP[beta S] may be unrelated to inhibition of G-protein-GTP interaction, but, instead, may be mediated via an extracellular site, common to all the nucleotides tested and perhaps via inhibition of the effects of endogenous/released ADP. The usefulness of GDP[beta S] as a tool in studying G-protein-GTP interactions in platelets is thus questionable.

Effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced arachidonate release and 5-hydroxytryptamine secretion in human platelets. Dependence of effects on agonist type and time of incubation with PMA

We have previously demonstrated synergistic potentiation of secretion by phorbol 12-myristate 13-acetate (PMA) and platelet agonists such as thrombin and the thromboxane mimetic, U46619, with short (less than 2 min) pre-incubations of PMA, despite inhibition of agonist-induced [Ca 2+] i mobilization and arachidonate/thromboxane release. In this study, the effect of PMA on 5-hydroxytryptamine secretion in relation to arachidonate/thromboxane B 2 release induced by collagen as well as the ‘weak agonists’, ADP, adrenaline and platelet-activating factor (PAF), was investigated using human platelet-rich plasma. Short incubations (10–30 s) with PMA (400 nM) before agonist addition caused an inhibition (60–100%) of 5-hydroxy[ 14C]tryptamine secretion and thromboxane B 2 formation in response to maximally effective doses of ADP (10 μM), adrenaline (10 μM) and PAF (0.5 μM) but potentiated collagen-induced 5-hydroxy[ 14C]tryptamine secretion and [ 3H]arachidonate/thromboxane release. However, a longer pre-incubation with PMA (5 min) caused a significant reduction (20–50%) in the extent of collagen-induced 5-hydroxy[ 14C]tryptamine secretion and thromboxane B 2 formation as seen earlier with thrombin, although collagen-induced [ 3]arachidonate release was still unaffected. Pretreatment of platelets with the cyclooxygenase inhibitor, indomethacin (10 μM), abolished 5-hydroxy[ 14C]tryptamine secretion in response to the weak agonists and reduced collagen (2.5–10 μg/ml) -induced secretion by 50–90%, depending on the collagen concentration. Addition of PMA (400 nM) 10 s before these agonists in indomethacin-treated platelets resulted in synergistic interactions between agonist and PMA leading to enhanced 5-hydroxy[ 14C]tryptamine secretion, although this was notably less than the synergism observed previously between thrombin and PMA or U46619 and PMA. The results suggest that the effect of short incubations with PMA on 5-hydroxytryptamine secretion induced by ‘thromboxane-dependent’ agonists, such as those examined in this study, is determined by the effect on agonist-induced thromboxane synthesis. However, when endogenous thromboxane synthesis is blocked, weak agonists as well as collagen can synergize with PMA at potentiating 5-hydroxytryptamine secretion, albeit to a weaker extent than thrombin or U46619. The results also suggest that PMA has differential effects on arachidonate release induced by collagen and thrombin.

Collagen stimulates [3H]inositol trisphosphate formation in indomethacin-treated human platelets.

The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.

Augmentation by Platelets of Granulocyte Aggregation in Response to Chemotaxins: Studies Utilizing an Improved Cell Preparation Technique

Considerable evidence exists to suggest roles for both platelets and granulocytes (PMNs) in pulmonary injury in shock. While believing that the major contributions of the two cell types are sequential, in vitro observations suggested that direct interactions between granulocytes and platelets might also amplify tissue damage. Using isotonic Percoll density gradients to isolate PMNs, we therefore studied the effect of deliberate platelet contamination on PMN aggregation. PMN aggregation in response to N-formyl-met-leu-phe or activated complement was enhanced by the presence of 1 platelet/PMN, an effect that became maximal at 16 platelets/PMN (p less than 0.01); large mixed aggregates were formed. Lysed, aspirinated, and indomethacin-treated platelets retained their augmentative capacity, as did platelets washed by gel filtration. The effect was not mimicked by the addition of histamine or serotonin to PMN preparations. None of these platelet preparations augmented lysosomal enzyme release. We conclude that platelets augment PMN aggregation, both by forming giant mixed PMN/platelet aggregates and also by producing a labile augmentative substance, the production of which may be independent of thromboxane synthesis. We propose that direct as well as sequential platelet/PMN interactions may be important in tissue injury in shock.

Augmentation by platelets of granulocyte aggregation in response to chemotaxins: studies utilizing an improved cell preparation technique

Considerable evidence exists to suggest roles for both platelets and granulocytes (PMNs) in pulmonary injury in shock. While believing that the major contributions of the two cell types are sequential, in vitro observations suggested that direct interactions between granulocytes and platelets might also amplify tissue damage. Using isotonic Percoll density gradients to isolate PMNs, we therefore studied the effect of deliberate platelet contamination on PMN aggregation. PMN aggregation in response to N-formyl-met-leu-phe or activated complement was enhanced by the presence of 1 platelet/PMN, an effect that became maximal at 16 platelets/PMN (p less than 0.01); large mixed aggregates were formed. Lysed, aspirinated, and indomethacin-treated platelets retained their augmentative capacity, as did platelets washed by gel filtration. The effect was not mimicked by the addition of histamine or serotonin to PMN preparations. None of these platelet preparations augmented lysosomal enzyme release. We conclude that platelets augment PMN aggregation, both by forming giant mixed PMN/platelet aggregates and also by producing a labile augmentative substance, the production of which may be independent of thromboxane synthesis. We propose that direct as well as sequential platelet/PMN interactions may be important in tissue injury in shock.

Inhibition of Platelet Aggregation by Protease Inhibitors. Possible Involvement of Proteases in Platelet Aggregation

Abstract The possible participation of proteases in human platelet aggregation was explored using various protease inhibitors and substrates. Protease inhibitors used included naturally occurring inhibitors of serine proteases and synthetic inhibitors that modify the active site of protease. Substrates used were synthetic substrates for the trypsin type as well as for the chymotrypsin type of protease. All these inhibitors and substrates inhibited platelet aggregation and serotonin release induced by ADP, collagen, epinephrine, or thrombin. In ADP- and epinephrine-induced platelet aggregation the second phase of aggregation was most efficiently inhibited. The inhibitors suppressed the formation of malondialdehyde during platelet aggregation. Release by aggregating agents of arachidonate and its metabolites from indomethacin-treated platelets as well as nontreated platelets was also inhibited. The inhibitors apperar to interact with stimulated platelets but not with unstimulated platelets. These observations suggest that the interaction of an aggregating agent with its platelet receptor activates a unique precursor serine protease that in turn activates platelet phospholipase to liberate arachidonic acid (the precursor of the potent platelet aggregating agent thromboxane A2) from platelet phospholipids.

Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation

The possible participation of proteases in human platelet aggregation was explored using various protease inhibitors and substrates. Protease inhibitors used included naturally occurring inhibitors of serine proteases and synthetic inhibitors that modify the active site of protease. Substrates used were synthetic substrates for the trypsin type as well as for the chymotrypsin type of protease. All these inhibitors and substrates inhibited platelet aggregation and serotonin release induced by ADP, collagen, epinephrine, or thrombin. In ADP- and epinephrine-induced platelet aggregation the second phase of aggregation was most efficiently inhibited. The inhibitors suppressed the formation of malondialdehyde during platelet aggregation. Release by aggregating agents of arachidonate and its metabolites from indomethacin-treated platelets as well as nontreated platelets was also inhibited. The inhibitors apperar to interact with stimulated platelets but not with unstimulated platelets. These observations suggest that the interaction of an aggregating agent with its platelet receptor activates a unique precursor serine protease that in turn activates platelet phospholipase to liberate arachidonic acid (the precursor of the potent platelet aggregating agent thromboxane A2) from platelet phospholipids.

Persistence of thromboxane A2-like material and platelet release-inducing activity in plasma.

During the incubation of arachidonic acid with platelet-rich plasma, a persistent activity appeared that caused the release of [14C]5-hydroxytryptamine from indomethacin-treated platelets. By the time-course of its appearance and disappearance, this release-inducing activity could be dissociated from prostaglandin endoperoxides and associated with thromboxane A2-like material. This material persists in plasma because of its continued production and increased stability.