Inhibitors of Na+/H+ exchange block stimulus-provoked arachidonic acid release in human platelets. Selective effects on platelet activation by epinephrine, ADP, and lower concentrations of thrombin

We have observed previously that removal of extraplatelet Na+ blocks platelet secretion of dense granule contents in response to epinephrine, ADP, and 0.004 unit/ml thrombin, all agents which must mobilize arachidonic acid for its subsequent conversion to cyclooxygenase products in order to elicit platelet secretion. The present studies demonstrate that removal of extraplatelet Na+ blocks arachidonic acid mobilization in response to epinephrine, ADP, and 0.004 unit/ml thrombin without altering arachidonic acid conversion to thromboxane A2. The data also provide several lines of evidence which suggest that the blockade of arachidonic acid release due to removal of extraplatelet Na+ is a manifestation of blockade of Na+/H+ exchange system. 1) There is a concentration-dependent effect of extraplatelet Na+ (EC50 congruent to 55 mM) on [3H]arachidonic acid release such that mobilization is observed when [Na+]o greater than [Na+]i. 2) Increasing extraplatelet [H+] (i.e. decreasing extraplatelet pH from pH 7.35 to 6.8) causes a concentration-dependent decline in stimulus-provoked [3H]arachidonic acid release. 3) Ethylisopropylamiloride and other potent 5-amino analogs of amiloride block [3H]arachidonic acid release with a potency that parallels their effects on Na+/H+ exchange in other cellular systems. None of the above manipulations alter primary aggregation induced by epinephrine, ADP, or 0.004 unit/ml thrombin, indicating that stimulus-receptor binding, subsequent exposure of fibrinogen receptors, and fibrinogen-mediated platelet-platelet cross-linking are not significantly inhibited by [3H]arachidonic acid release in response to greater than 0.1 unit/ml thrombin, a stimulus that can elicit platelet secretion in the absence of products of the cyclooxygenase pathway. Therefore, Na+/H+ exchange may selectively modulate arachidonic acid mobilization in response to the so-called "weak agonists," agonists that require this mobilization to effect vigorous platelet aggregation and dense granule secretion.     

A potent inhibitor of platelet activating factor from the saliva of the leech Hirudo medicinalis.

Leech saliva is shown to contain protein platelet aggregation inhibitors and a range of selective low molecular weight (LMW) aggregation inhibitors. Gel filtration on Bio-Gel P-2 (cut-off kDa) yields a protein fraction (Fr. I) and three LMW fractions. Fr. I inhibits aggregation induced by collagen, ADP, epinephrine and arachidonic acid. Of all the fractions, only one, Fr. II (LMW) specifically inhibits aggregation induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine). Fr. II also inhibits thrombin-induced platelet aggregation. Fr. III inhibits aggregation induced by ADP, epinephrine and arachidonic acid, and Fr. IV only that induced by arachidonic acid. Fr. II also inhibits PAF- and thrombin-induced thromboxane generation in platelets, but does not inhibit arachidonic acid-induced thromboxane generation. Efforts to separate the anti-PAF from the anti-thrombin activity have been unsuccessful. The inhibition may therefore be due to a single inhibitor, though it may also be due to several inhibitors. Fr. II also inhibits superoxide anion production in formyl Met-Leu-Phe (fMLP)- and ionophore 23187- stimulated neutrophils. This may be due to the inhibition of the effects of PAF generated within the cell. Preliminary results suggest that the Fr. II inhibitor(s) is (are) amphipathic. The interaction of platelets with PAF and their interaction with the inhibitor(s) are mutually exclusive, and the inhibition may be competitive.     

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani

Three acidic phospholipases A₂ from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 M). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A₂ with platelet rich plasma. Parabromophenacyl bromide modified PLA₂ enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers

Effects of inhaled nitric oxide (NO) on human platelet function are controversial. It is uncertain whether intraplatelet cGMP mediates the effect of inhaled NO on platelet function. We investigated the effect of 30 ppm inhaled NO on platelet aggregation and plasma and intraplatelet cGMP in 12 subjects. We performed platelet aggregation studies by using a photooptical aggregometer and five agonists (ADP, collagen, epinephrine, arachidonic acid, and ristocetin). During inhalation, the maximal extent of platelet aggregation decreased by 75% with epinephrine (P < 0.005), 56% with collagen (P < 0.005), and 20% with arachidonic acid (P < 0.05). Responses to ADP (8% P > 0.05) and ristocetin (5% P > 0.05) were unaffected. Platelet aggregation velocity decreased by 64% with collagen (P < 0.005), 60% with epinephrine (P < 0.05), 33% with arachidonic acid (P < 0.05), and 14% with ADP (P > 0.05). Plasma cGMP levels increased from 2.58 +/- 0.43 to 9.99 +/- 5.57 pmol/ml (P < 0.005), intraplatelet cGMP levels were unchanged (means +/- SD: 1.96 +/- 0.58 vs. 2.71 +/- 1.67 pmol/109 platelets; P > 0.05). Inhaled NO inhibits platelet aggregation via a cGMP independent mechanism.     

Demonstration of PGl2 production by isolated perfused rat lungs with platelet aggregation test

Platelet aggregation test was used for PGl₂ measurements. The use of 6 % CO₂ in air stabilized human platelet rich plasma (PRP) so that it could be used for up to 7 hours in these measurements. The PGl₂ caused inhibition of aggregation in response to ADP was concentration dependent in the range of 0.5 ng/ml to 50 ng/ml. Isolated perfused rat lungs released spontaneously 190 ng/min PGl₂ and about 3 % of infused arachidonic acid potassium salt (equivalent to 25 μg/min arachidonic acid) was converted to PGl₂.     

Characterization of the normal bovine platelet aggregation response

1. Bovine platelets are more sensitive to stimulation by platelet activating factor (PAF) than adenosine-di-phosphate (ADP) or thrombin. 2. While epinephrine, arachidonic acid and serotonin are ineffective by themselves as aggregatory stimulants of bovine platelets they enhance the aggregation response of other platelet agonists. 3. There is no correlation between thromboxane A2 production and release and the extent of platelet aggregation in bovine platelets. 4. The dependence of bovine platelet aggregation on a phospholipid pathway and calcium mobilization is indicated.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56-98%, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC50 values for E. vespertilio and C. ambiguus estimated to be 20.4 microl (1.855 mg/ml) and 8.34 microl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.     

Effects of acrolein on human platelet aggregation

Acrolein, a component of tobacco smoke, potentiated platelet aggregation and increased thromboxane A2 (TXA2) formation caused by thrombin and arachidonic acid (AA). Acrolein produced these effects at concentrations in the range 50-5000 microM. Acrolein had no effect on platelet responses to ADP, epinephrine, collagen or the ionophore A23187. Acrolein increased the mobilization of [3H]arachidonic acid from prelabelled platelets in response to thrombin and arachidonic acid. The increased availability of substrate could partly explain the enhanced production of TXA2 and increased aggregation observed in the presence of acrolein. These findings could provide an explanation for the increased incidence of vascular disease in cigarette smokers.     

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Effects of N-acetylglucosamine and platelet inhibitors on the synergistic interaction of platelets and aggregating agents in the presence of wheat germ agglutinin

Low concentrations of wheat germ agglutinin (4 micrograms/ml) have been shown to act synergistically to induce platelet aggregation with epinephrine, collagen, arachidonate and ionophore A23187. Aggregation ceased on the addition of the haptenic sugar N-acetylglucosamine at any time following the onset of aggregation with these agonists and a small degree of disaggregation was observed during the reversible first wave with the biphasic aggregating agents epinephrine and ADP. Cyclooxygenase inhibitors such as indomethacin and aspirin blocked the second wave of aggregation with the biphasic aggregating agents epinephrine and ADP but a synergistic response continued to be shown with the first wave in the presence of these inhibitors. Release of [14C]serotonin and the mobilization of [3H]arachidonate by epinephrine and collagen were markedly stimulated in the presence of wheat germ agglutinin but there was no increase of either radiolabel in the case of ADP. Platelet shape change, but not aggregation, occurred with low levels of wheat germ agglutinin and the synergistic response with ADP, collagen or ionophore A23187 occurred without further shape change. Wheat germ agglutinin did not affect the basal or stimulated levels of cyclic AMP. The membrane fluidity of platelets was not affected by the lectin or by thrombin as shown by the lack of change in fluorescence polarization with diphenylhexatriene. It is suggested that the binding of wheat germ agglutinin to the platelet surface induces platelet activation by mechanisms similar to those of other agonists and that it may affect the distribution of membrane-bound Ca2+ by a reversible perturbation of the platelet membrane.     

Postprandial hyperlipemia inhibits platelet aggregation without affecting prostanoid metabolism

The objective of this work was to characterize changes in platelet aggregability during postprandial hypertriglyceridemia with special emphasis on arachidonic acid metabolism. Ten healthy young men consumed 100 g fat after a fasting period of 12 hr. In-vitro platelet aggregation induced by ADP and collagen was measured at 0, 3, 5, and 9 hours after the fat intake. The major arachidonic acid metabolites, 12-hydroxyeicosatetraenoic acid (12-HETE), thromboxane A2 (TXA2), prostaglandin F2 alpha (PGF2a), and prostaglandin E2 (PGE2) produced during collagen-induced platelet activation were quantified by gas chromatography/mass spectrometry. A significant decrease in platelet aggregability induced by both ADP and collagen was detected during the postprandial hyperlipemia. No significant changes could be found in the prostanoid pattern of collagen activated platelets. There was no correlation between the degree of the inhibition of platelet aggregation and the relative or absolute increase of triglyceride-levels in the plasma during the postprandial hyperlipemia.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP

The capacity of epinephrine alone and the combination of low dose epinephrine and ADP to support the binding of fibrinogen to washed human platelets has been examined, 125I-Fibrinogen was bound to epinephrine-stimulated platelets, but 90 min were required to achieve maximal binding at 22 degrees C in contrast to 20 to 30 min with ADP. The overall rate of interaction appeared to reflect the slow binding of fibrinogen to epinephrine-stimulated platelets as opposed to the rate of stimulation of the cell. Divalent ions were required for binding of fibrinogen to epinephrine-stimulated platelets, and both calcium and magnesium supported binding with a prolonged time course. Fibrinogen binding was maximally supported by 20 to 30 microM epinephrine. The combination of low dose epinephrine (5 microM) and low dose ADP (0.5 microM), which acted synergistically to induce platelet aggregation, supported the rapid (10 min) binding of fibrinogen to platelets. With 4 microM epinephrine, more fibrinogen bound per platelet at all ADP doses than with ADP alone. With all the stimuli, saturable binding of fibrinogen to the platelet was observed, and Scatchard plots were linear, yielding very similar apparent association constants. The number of molecules bound per cell was stimulus-dependent, with 30 microM epinephrine inducing the binding of fewer fibrinogen molecules per cell (mean = 20,400) than 10 microM ADP (mean = 35,900) or the combination of 5 microM epinephrine + 0.5 microM ADP (mean = 43,600). The participation of endogenous ADP in fibrinogen binding to epinephrine-stimulated platelets was suggested since enzymes which remove ADP, apyrase, and creatine phosphate/creatine phosphokinase, and the ADP analogue, 2-chloroadenosine, completely inhibited the binding of fibrinogen to the platelet.     

Effects of N-acetylglucosamine and platelet inhibitors on the synergistic interaction of platelets and aggregating agents in the presence of wheat germ agglutinin

Low concentrations of wheat germ agglutinin (4 μg/ml) have been shown to act synergistically to induce platelet aggregation with epinephrine, collagen, arachidonate and ionophore A23187. Aggregation ceased on the addition of the haptenic sugar N-acetylglucosamine at any time following the onset of aggregation with these agonists and a small degree of disaggregation was observed during the reversible first wave with the biphasic aggregating agents epinephrine and ADP. Cyclooxygenase inhibitors such as indomethacin and aspirin blocked the second wave of aggregation with the biphasic aggregating agents epinephrine and ADP but a synergistic response continued to be shown with the first wave in the presence of these inhibitors. Release of [¹⁴C]serotonin and the mobilization of [³H]arachidonate by epinephrine and collagen were markedly stimulated in the presence of wheat germ agglutinin but there was no increase of either radiolabel in the case of ADP. Platelet shape change, but not aggregation, occurred with low levels of wheat germ agglutinin and the synergistic response with ADP, collagen or ionophore A23187 occurred without further shape change. Wheat germ agglutinin did not affect the basal or stimulated levels of cyclic AMP. The membrane fluidity of platelets was not affected by the lectin or by thrombin as shown by the lack of change in fluorescence polarization with diphenylhexatriene. It is suggested that the binding of wheat germ agglutinin to the platelet surface induces platelet activation by mechanisms similar to those of other agonists and that it may affect the distribution of membrane-bound Ca²⁺ by a reversible perturbation of the platelet membrane.     

Effects of repeated oral doses of fenflumizole on platelet aggregation and thromboxane formation in man ex vivo

Anti-platelet effects of fenflumizole, a new cyclo-oxygenase inhibitor, were studied in man ex vivo. Fenflumizole was given to male volunteers at the oral doses of 25, 50 or 100 mg per day, each dose for a period of seven days. The formation of thromboxane B2 (TXB2) during whole blood clotting, platelet aggregation induced by arachidonic acid and ADP, the formation of TXB2 during aggregation as well as serum concentration of fenflumizole were measured repeatedly during drug administration and for a fortnight after drug discontinuation. TXB2 formation during whole blood clotting was decreased dose-dependently by fenflumizole. The degree of inhibition of TXB2 formation was proportional to fenflumizole concentration in serum within each individual. The lag phase of platelet aggregation induced by arachidonic acid was prolonged and the formation of TXB2 during aggregation decreased by fenflumizole. No total inhibition of either TXB2 synthesis or platelet aggregation was caused by the fenflumizole doses used. The results show that the degree of inhibition of platelet thromboxane forming capacity by repeated doses of fenflumizole is closely related to the concentration of the drug in blood. Platelet aggregation however is less sensitive to changes in fenflumizole levels and cannot be assessed solely on the basis of cyclo-oxygenase activity.     

Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Dependence of the thrombocyte aggregation reaction on plasma fibronectin and the tripeptide arginyl-glycyl-asparagilin

If was shown that the addition of fibronectin antibodies exerted the inhibition of platelet aggregation. The tripeptide RGD inhibited the platelet aggregation induced by the same agents (ADP, epinephrine, thrombin, collagen) both in blood plasma and in suspension of washed platelets.