Effect of op 1206, a prostaglandin E1 derivative, on guinea-pig platelet functions

OP 1206, 17(S)-methyl-ω-homo-trans-Δ²-prostaglandin E₁, inhibited guinea-pig platelet aggregation induced by ADP, collagen, A 23187, arachidonic acid, labile aggregation stimulating substances (LASS) and thromboxane A₂ (TXA₂)-like substance, and platelet adhesiveness to a glass bead column. The potency of inhibition was 10–16 times stronger than that of prostaglandin E₁ (PGE₁). ADP-induced platelet aggregation was notably disaggregated by the addition of OP 1206 after induction of aggregation. The release of ATP and ADP from guinea-pig platelets induced by collagen was suppressed by OP 1206, of which potency was 9–10 times stronger than that of PGE₁. OP 1206 and PGE₁ increased guinea-pig platelet cyclic AMP levels, and the increased levels were augmented by the pretreatment with theophylline. OP 1206 and PGE₁ inhibited synthesis of guinea-pig platelet malondialdehyde (MDA) induced by thrombin but not by arachidonic acid. This inhibition was released by exogenous calcium. OP 1206 and PGE₁ showed no influence on synthesis of radioactive TXA₂ (measured as a stable form, TXB₂) from [¹⁴C] arachidonic acid. From these results, increased levels of platelet cyclic AMP by OP 1206 as well as PGE₁ may exert their action on platelet functions.     

Discrimination between platelet prostaglandin receptors with a specific antagonist of bisenoic prostaglandins

Platelet aggregation and secretion induced by PGG₂ and by analogues of PGH₂ and PGE₂ were inhibited by NO164. Inhibition was apparently competitive (K_i 20 μM). Responses to ADP, vasopressin and arachidonic acid were unaffected. Inhibition of ADP-induced platelet aggregation by PGD₂ and PGE₂ (but not by PGE₁) was antagonised by NO164.     

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E₁ (PGE₁) on platelets is mediated through the PGE₁ receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE₁ and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 gm. The time course of the extension length (Dp, in μg) of the platelets in response to aspiration with a negative pressure (ΔP) of 5 cm H₂ O (ΔP × Rp = 0.15 dynes/cm) was analyzed. PGE₁ treatment (0.1 μM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE₁ -treated) / Dp/Rp (control), was significantly reduced to 0.90 ± 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE₁ -treated platelets had a higher [cAMP]_i than controls. Platelets treated with PGE₁ or dbcAMP showed a reduced [Ca²⁺]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE₁ and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]_i and low [Ca²⁺]_i after PGE₁ treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.     

Effect of cobra venom phospholipase A2 on platelet aggregation in comparison with those produced by arachidonic acid and lysophophatidylcholine

Cobra venom phospholipase A₂ induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B₂ formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE₁ and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B₂ formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A₂ with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B₂ formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A₂ is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.     

The influence of glutathione and other thiols on human platelet aggregation

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC₅₀=0.61 ± 0.05 mM) was greater than that by cysteine (IC₅₀=13 ± 1 mM) or 6-mercaptopurine (IC₅₀=5.4 ± 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.     

Selective antagonism of prostaglandin (PG) E1, PGD2 and prostacyclin (PGI2) on human and rabbit platelets by DI-4-phloretin phosphate (DPP)

The inhibition of human and rabbit platelet aggregation by prostaglandin (PG) E₁, PGD₂ and prostacyclin (PGI₂) was examined to determine if the three PGs inhibit platelets via a common receptor. Di-4-phloretin phosphate (DPP) was found to antagonise PGD₂, but not PGE₁ or PGI₂ on human platelets. In contrast, on rabbit platelets, DPP antagonised PGI₂ and PGE₁ but not PGD₂. The results suggest that there are two distinct types of PG receptors on human and rabbit platelets activated either by prostacyclin or PGD₂.     

Cyclic AMP independent inhibition of platelet aggregation by prostaglandin E1 is mediated through factor Xa

Normal plasma exposed to an insolublized ω-NH2-hexyl-agarose derivative of PGE₁ inhibits ADP-induced platelet aggregation. To define the plasma macromolecule responsible, we tested plasma congenitally deficient in coagulation factors for this property. Only plasma deficient in factor X failed to inhibit platelet aggregation under these conditions. The inhibition could be restored by reconstituting the defective plasma with bovine or human factor X. Purified factor X or Xa exposed to insolubilized PGE₁ also inhibited ADP-induced platelet aggregation. Factor Xa could reverse the inhibition of altered factor X but the reverse was not true suggesting that factor Xa was the responsible component. This conclusion was further supported by the ability of heparin to reverse the inhibitory effect of altered factor Xa when the anti-coagulant was added simultaneously to platelets. The results were not due to the release of PGE₁ from the insoluble derivative. Factor Xa may have a heretofore unrecognized effect on modulating platelet functions.     

Influence of hypercapnia and hypoxia on rabbit platelet aggregation

The influence of changes in pCO₂, pH and pO₂ on the aggregation of rabbit blood platelets was studied , with emphasis on hypercapnia, acidocis and hypoxia. Hypercapnia combined with acidosis caused a reduction in rabbit platelet aggregation, as induced by collagen, thrombin and ADP; the effect being most pronounced with collagen and smallest with ADP. Hypoxia reduced thrombin induced platelet aggregation, but had no effect on ADP and collagen induced aggregation. Synergistic activation of rabbit platelets, as induced by the addition of serotonin to platelet rich plasma together with collagen or ADP, seemed to be equally sensitive to changes in pCO₂ and pH as activation by the individual agents, and insensitive to changes in pO₂.     

Response of newborn and adult sheep to pyrogens: relation between fever and brain eicosanoid changes

We investigated whether the weak febrile response to pyrogens in newborns is due to a diminished activation of the putative pyrogen mediator, prostaglandin (PG)E₂. Indwelling cannulas in the third ventricle of lambs (age, 5–31 days) and adult ewes were used to collect cerebrospinal fluid (CSF) for radioimmunoassay of PGE₂. Intravenous (i.v.) endotoxin caused a smaller increase in body temperature but a larger increase in CSF PGE₂ in lambs compared to adults. PGE₂ by intracarotid infusion raised body temperature in 5 of 7 trials in 3 lambs and in 4 of 4 trials in 1 adult. Endotoxin given intracerebroventricularly (i.c.v.) induced a rise in temperature and CSF PGE₂ in the lamb but, in the adult, these responses were delayed and smaller. Interleukin-1 i.c.v. and PGE₂ i.c.v. were weak pyretic agents at both ages. We conclude that the lamb's diminished febrile response to endotoxin i.v. is not caused by a lesser rise in CSF PGE₂; rather it may be due, at least in part, to reduced responsiveness to this putative mediator. Regardless of age, the sheep differs from other species in that pyrogen/PGE₂ coupling occurs primarily at a site in brain that is better accessible from blood than CSF.     

Role of intracellular signaling events in ADP-induced platelet aggregation.

Human platelets express two distinct G protein-coupled ADP receptors, one coupled to phospholipase C through Gq, P2Y1, and the other to inhibition of adenylyl cyclase through Gi, P2TAC. We have recently shown that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Previous studies have tested whether ADP causes a decrease in the basal cAMP level and this reduction promotes platelet aggregation, but did not study the effect of decreased cAMP levels when the Gq pathway is selectively activated. Since we are now aware that platelet aggregation requires activation of two receptors, we investigated whether the function of P2TAC receptor activation, leading to inhibition of platelet adenylyl cyclase, could be replaced by direct inhibition of adenylyl cyclase, when Gq pathway is also activated, a possibility that has not been addressed to date. In the present study, we supplemented the P2Y1 mediated Gq signaling pathway with inhibition of the platelet adenylyl cyclase by using SQ22536 or dideoxyadenosine, or by selective activation of the alpha2A adrenoceptors with epinephrine. Although SQ22536, dideoxyadenosine, and epinephrine reduced the cAMP levels, only epinephrine could mimic the P2TAC receptor mediated signaling events, suggesting that reduction in basal cAMP levels does not directly contribute to ADP-induced platelet activation. Adenosine-5'-phosphate-3'-phosphosulfate, a P2Y1 receptor antagonist, completely blocked ADP-induced inositol 1,4,5-trisphosphate and inositol 1,3.4-trisphosphate formation suggesting that P2TAC-mediated activation of Gi (or other G proteins) does not activate phospholipase C. These results suggest that a signaling event downstream from Gi, independent of the inhibition of platelet adenylyl cyclase, contributes to alphaIIb beta3 activation.     

Effects of prostaglandin E2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus

The present study attempts to elucidate the possible role of adenosine 3′,5′-monophosphate (cAMP) and prostaglandin E₂ (PGE₂) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH-RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P₂ membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 μg/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step-wise increment in several doses of PGE₂ (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE₂ induced significant (P<0.01) increments in LH-RH release at doses of 5.68 × 10⁻⁷, 5.68 × 10⁻⁶, and 5.68 × 10⁻⁵ M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH-RH release was also obtained; forskolin and cholera toxin significantly (P<0.01) stimulated LH-RH release at doses of 1 × 10⁻⁴ and 5.4 × 10⁻¹⁰ M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium. An intermittent infusion of dibutyryl cAMP (dbcAMP: 1 × 10⁻⁷ M, 10-min on, 20-min off) resulted in rhythmic LH-RH release from median eminences superfused in vitro. In separate experiments, to examine adenylate cyclase activity, P₂ membrane fractions from the mediobasal hypothalamus were preincubated with appropriate test agents. Adenylate cyclase reaction was initiated by adding adenosine triphosphate. After a 15-min incubation, the reaction was terminated by boiling, the supernatant recovered and subjected to cAMP determination by RIA. The following results were obtained: (1) in vivo E priming of ovariectomized animals significantly increased basal adenylate cyclase activity of P₂ membrane preparations as compared to those from unprimed rats; (2) known adenylate cyclase activators, such as forskolin and cholera toxin clearly produced a dose-related increase in cAMP production; and (3) PGE₂ at the concentration of 5.68 × 10⁻⁶ M stimulated cAMP production. It appears that cAMP and PGE₂ may be involved in the activation of the LH-RH neural apparatus. It is tempting to postulate that PGE₂ may stimulate adenylate cyclase to increase intracellular cAMP levels which, in turn, trigger the release of LH-RH from the median eminence nerve terminals.     

SQ 22536, an adenylate-cyclase inhibitor, prevents the antiplatelet effect of dazoxiben, a thromboxane-synthetase inhibitor

This study shows that dazoxiben, a selective inhibitor of thromboxane A2-synthetase in human platelets, inhibited arachidonic acid-induced platelet aggregation in platelet-rich plasma samples from four out of 16 healthy volunteers. In these four "responder" samples, the anti-aggregating effect of dazoxiben was prevented by the compound SQ 22536, a 9-substituted adenine analogue, endowed with an inhibitory activity on adenylate-cyclase. The compound SQ 22536 also counteracted the antiaggregating effect of prostaglandin D2, a known activator of platelet adenylate-cyclase. When platelet thromboxane A2-synthetase was blocked by dazoxiben, a marked increase of prostaglandin D2 was concomitantly observed both in "responder" and "non responder" samples. The compound SQ 22536 blunted the increase in platelet cAMP caused by either dazoxiben and sodium arachidonate or prostaglandin D2. It is suggested that the antiaggregating effect of dazoxiben is mediated by newly synthesized prostaglandin D2. The latter acts by stimulating adenylate-cyclase and increasing cAMP levels. The compound SQ 22536 prevents both phenomena. In "non responder" samples some factors--still to be defined--might counteract similarly to the compound SQ 22536 the antiaggregating activity of PGD2.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production

We studied the effects of picotamide (N,N′ bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B₂ (TxB₂) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/1) inhibited platelet aggregation, the release of ATP and TxB₂ production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/1) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB₂ production both under stirring and non-stirring conditions, whereas the pure thromboxane A₂ receptor antagonist BM13177 (0.5 mmol/1) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB₂ production, whereas BM13177 inhibits the potentiation of TxB₂ production due to TxA₂/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA₂/PE antagonism; (ii) inhibition of thromboxane synthase.     

Effect of EDTA and PGE1 on beta-thromboglobulin liberation from platelets

The aim of this study was to evaluate the effect of PGE₁ and EDTA on liberation of β-thromboglobulin (βTG) from platelets in vitro. Liberation of BTG was followed in citrated blood at room temperature for 120 minutes after venesection. PGE₁ reduced βTG liberation, and maximal inhibition was attained by concentrations greater than 2 × 10⁻⁶M. EDTA induced the efflux of βTG. This EDTA-induced efflux was delayed but not prevented by PGE₁ and by citrate; it was not found at 0–4°C. Therefore the use of EDTA to prevent βTG liberation during sampling for in vitro or in vivo studies depends heavily on modifying factors such as PGE₁ and low temperature, and on the time taken to process samples. Its effectiveness must be in some doubt where the platelets may be sufficiently stimulated to overcome these modifying influences, or where handling of samples is less than optimal.     

Thromboxane synthase inhibition causes re-direction of prostaglandin endoperoxides to prostaglandin D2 during collagen stimulated aggregation of human platelet rich plasma

Prostanoid synthesis and release during collagen-induced aggregation of human platelet rich plasma (PRP) was studied using a novel gas chromatography/mass spectrometry assay technique. Aggregation was associated with the production of mainly thromboxane A₂ (TXA₂), measured as TXB₂, and smaller amounts of the prostaglandins (PGs) D₂, E₂ and F₂. UK 37,248 inhibited T₁XB₂ formation by >95% and increased the production of PGD₂, PGE₂ and PGF₂ twenty-fold. The relative amounts of these three prostanoids were not changed by UK 37,248. Even though high concentrations of PGD₂ were formed, aggregation was not inhibited. In contrast, flurbiprofen inhibited aggregation, demonstrating that platelet aggregation produced by this concentration of collagen is cyclooxygenase dependent. These results support the proposal that the prostaglandin endoperoxides can induce aggregation alone, irrespective of the amount of PGD₂ that is produced.     

Effects of phenacetin and its metabolite p-phenetidine on COX-1 and COX-2 activities and expression in vitro

The present study was aimed to test the possible cyclooxygenase (COX)-1/COX-2 selectivity of the old analgesic drug phenacetin and its metabolite p-phenetidine, which exhibits high renal toxicity. Paracetamol (acetaminophen), the main metabolite of phenacetin with low renal toxicity, and indomethacin were selected as reference compounds. Collagen-stimulated platelet thromboxane B₂ (TxB₂) production and phorbol 12-myristate-13-acetate (PMA)-induced neutrophil prostaglandin E₂ (PGE₂) synthesis were used as indicators for COX-1 and COX-2 activity, respectively. Phenacetin was even less potent than paracetamol to reduce the production of both TxB₂ and PGE₂, and no clear preference for either of the COX-enzymes was seen. p-Phenetidine was a more potent inhibitor, already at nanomolar level, of the synthesis of these prostanoids than indomethacin and showed some preference to COX-2 inhibition. Somewhat higher, micromolar, concentrations of p-phenetidine also reduced COX-2 expression in neutrophils. We suggest that the very potent inhibitory activity of p-phenetidine on PGE₂ synthesis combined with the reduction of COX-2 expression could explain the renal papillary necrosis in phenacetin kidney.     

Involvement of calmodulin in platelet reaction

Penothiazines, known as selective inhibitors of calmodulin, completely inhibited platelet aggregation and secretion induced by ADP, collagen, epinephrine, thrombin or calcium ionophore. They also completely inhibited aggregation induced by exogenous arachidonate (AA) or a mixture of thromboxane A₂ and prostaglandin endoperoxides (TxA₂/PG G₂,H₂). Also, in the presence of these calmodulin inhibitors, the release of AA from platelet phospholipids (PL) was dosedependently inhibited in stimulated platelets. These observations suggest that in platelet reaction, calmodulin is involved in at least two different steps of the reaction: activation of phospholipases and contraction of platelet actomyosin after the formation of TxA₂.     

Thromboxane A2 and the endoperoxides mediate canine platelet activation

With canine platelets, arachidonate consistently induces a shape change and potentiates the response to a submaximal ADP stimulus even though it does not generally induce aggregation. We have determined that TXA₂ and possibly the endoperoxides are responsible for these effects based on the following data: (i) indomethacin (14uM) blocks both actions of arachidonate, (ii) imidazole (5uM) partially blocks both, and (iii) the endoperoxide analog, U-46619, produces effects similar to arachidonate, whereas other stable metabolites of arachidonate (PGE₂, PGF₂, PGD₂, PGI₂, 6-keto-PGF₁ and TXB₂) do not. Specifically, PGI₂ and PGD₂ inhibit ADP-induced aggregation; low concentrations of PGE₂ and PGF₂ facilitate secondary aggregation; and the other metabolites are relatively inactive. With the exception of PGF₂ , these activities are qualitiatively identical to those observed with human platelets. A decreased sensitivity of canine platelets to TXA₂ and the endoperoxides is advanced as the explanation of their diminished responsiveness to arachidonate relative to other species.     

Response of cyclic nucleotides to stimulation by prostaglandin E1 and 5-hydroxytryptamine in stored human platelets

Levels of cyclic nucleotides and their response to stimulation by prostaglandin E₁ (PGE₁) and 5-hydroxytryptamine were studied in human platelets stored for up to 48 hr at 22°C and 4°C. During storage at 22°C for 48 hr platelet cAMP levels declined gradually by approximately 20% while cGMP registered a 50% decrease. Levels of both cyclic nucleotides remained relatively stable in platelets stored at 4°C. Compared to prestorage controls 22°C stored platelets showed enhanced PGEl-stimulated cAMP production but had a depressed 5-HT-induced cGMP response. The latter was increased in 4°C stored platelets. Kinetic studies indicated that the enhanced PGE₁-stimulated cAMP production was due to an increase in the maximal capacity to produce cAMP while the depressed 5-HT-induced cGMP response was due to a decrease in the sensitivity to 5-HT of platelets stored at 22°C. Conversely,an increased sensitivity was responsible for the enhanced cGMP response to 5-HT stimulation in platelets stored at 4°C. These findings of our experiments are in accord with the documented changes in aggregability of stored platelets.