Human plasma transforms prostacyclin (PGI2) into a platelet antiaggregatory substance which contracts isolated bovine coronary arteries

Prostacyclin (PGI₂) incubated in Human Platelet Rich Plasma (PRP); in Platelet Poor Plasma (PPP) or in Krebs-Ringer-Bicarbonate (KRB) during different periods of time on contractions of bovine coronary arteries and on the ADP platelet aggregative capacity of human PRP, were explored. It was documented that incubates in PRP or in PPP retain an antiaggregatory activity at higher levels and during a longer time than in KRB. On the other hand, PGI₂ incubates in KRB exhibited only a relaxing activity on isolated bovine coronary arteries, whereas when incubated in PRP or in PPP presented a biphasic influence. The initial effects (evoked by incubates of 30 minutes) were distinctly relaxing but those obtained with later incubates (60–150 minutes) stimulated clearly the resting basal tone of the arteries. The possibility that the human plasma might have an enzyme(s) able to transform prostacyclin into a more stable material with human antiaggregatory platelet function and bovine coronary contracting capacity is discussed.     

Release of prostaglandin I2-like activity from the rat aorta: Effect of captopril,furosemide, and sodium

The effect of captopril, furosemide, indomethacine and intake of sodium on the production of PGI₂-like material was studied in the rat aorta. Release of PGI₂-like material from these vessels was estimated by its ability to inhibit ADP-induced vessels was estimated by its ability to inhibit ADP-induced platelet aggregation. Pretreatment with indomethacin (15 mg/kg/day) reduced the capacity of the aorta to release PGI₂-like material. Pretreatment with captopril (10 mg/kg/day) had no effect. Intravenous furosemide (60 μg/ml plasma volume) increased the capacity of the aorta to inhibit by 28% (p<.025). The inhibitory capacity of aorta removed from rats on a low sodium diet did not differ from those on a high sodium diet. We conclude that the action of furosemide in reducing vascular tone may be related to stimulation of PGI₂ synthesis in blood vessels whereas the effect of captopril and sodiumin in reducing vascular tone may involve a mechanism unrelated to PGI₂ synthesis or may involve the synthesis of a prostaglandin other than PGI₂.     

Microsomal preparations of normal bovine iris-ciliary body generate prostacyclin-like but not thromboxane-A2-like activity

Indomethacin-treated bovine iris-ciliary body microsomes (IBIM) have been studied for their ability to convert PG endoperoxides into either thromboxance-A₂ (TxA₂)-like or prostacyclin (PGI₂)-like activity. The biological activity of the ocular tissue microsomes were compared with either indomethacin-treated human platelet microsomes (for TxA₂-like activity) or rabbit aorta microsomes (for PGI₂-like activity) under appropriate incubation conditions. No evidence could be found for the formation of TxA₂-like activity from PG endoperoxides by the IBIM. In contrasts, when the IBIM were incubated with PGH₂ for 1 min at 22°C without cofactors, PGI₂-like activity was produced, causing profound relaxation of the isolated dog coronary artery preparation without contracting the rabbit aorta and inhibiting arachidonic acid-induced platelet aggregation. Equivalent quantities of boiled IBIM failed to aleter the biological activity of PGH₂ under identical conditions. Tranylcypromine (500 μg/ml) completely abolished the appearance of PGI₂-like activity. Furthermore, the PGI₂-like activity found was stable for 10 min at 22°C at pH 8.5 but completely lost under similar conditions at pH 5.5. It is concluded than microsomal preparations of normal bovine iris-ciliary body can synthesize PGI₂-like activity in substantial amounts but not TxA₂-like activity.     

On the multiplicity of platelet prostaglandin receptors I. Evaluation of competitive antagonism by aggregometry

Methods for the evaluation of competitive interactions at receptors associated with platelet activation and inhibition using aggregometry of human PRP have been developed. The evidence supports the suggestion that PGE₁ and PGI₂ share a common receptor for inhibition of platelet reactivity, but only a portion (if any) of the aggregation stimulation associated with PGE₂ is the result of PGE₂ binding (without efficacy) to this receptor. PGE₂ (@.3–20 μ ) is an effective antagonist of PGE₁, PGI₂, producing a shift of about one order of magnitude in the IC₅₀-values obtained from complete aggregation inhibition dose response curves. The antagonism of PGD₂ inhibition is particularly notable, 80 n PGE₂ levels are detectable. This and other actions of PGE₂ indicate another platelet receptor for PGE₂. PGE₁ acts at both the PGE₂ and PGI₂ receptor. Other substances showing PGI₂-like actions only at high doses (1–30 μ ), display additive responses with PGI₂ indicative of decreased affinity for the I₂/E₁ receptor and the absence of PGE₂-like aggregation stimulation activity.PGI₂ methyl ester has intrinsic inhibitory action not associated with in situ ester hydrolysis. The methyl ester is dissaggregatory showing particular specificity for inhibition of release and second wave aggregation.     

Production of prostacyclin-like substance in stroke-prone and stroke-resistant spontaneously hypertensive rats

Activities of aortae to produce prostaglandin (PG) I₂-like substance in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR) and normotensive control rats from the Wistar-Kyoto (WK) colony were compared. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and the amount produced was estimated by comparison of its anti-aggregatory activity with that produced by known amounts of the sodium salt of synthetic PGI₂. Before the development of stroke, amounts of this substance generated in SHRSP and SHRSR were significantly higher than those in WK rats (p<0.01 and p<0.02, respectively). Remarkably reduced capacity to generate PGI₂-like substance was observed in some SHRSP after the development of stroke.     

Prostacyclin production of rat aorta “in vitro” is increased by the combined action of dipyridamole plus pentoxifylline

The present study evaluates the effect of dipyridamole and pentoxifylline, individually and in combination, on PGI₂-like production and arachidonic acid metabolism of rat aorta “in vitro”. Pentoxifylline 100 μM and dipyridamole 92 and 184 μM increased PGI₂-like activity, as measured by the platelet aggregation inhibitory capacity of the aortic ring incubates, by 71%, 46% and 60% respectively; a greater increase in PGI₂-like activity was observed with the combination of the drugs than when they were used separately. This effect was observed even at the lowest doses assayed. In fact, dipyridamole 9.2 μM plus pentoxifylline 1 μM increased the PGI₂-like activity by 30% while the individual increase was 4.5% and 10.6% respectively. To obtain more information on the effect of the dipyridamole-pentoxifylline combination on arachidonic acid metabolism, arteries were incubated with (1-¹⁴C) arachidonic acid, and the 6-keto-PGF_1α and PGE₂ quantified. Dipyridamole 92 μM plus pentoxifulline 1 and 10 μM increased 6-keto-PGF_1α and PGE₂ production by about 30% and 48% respectively while combination with pentoxifylline 100 μM increased the 6-keto-PGF_1α 76.5% and the PGE₂ 50%. The possible biological effect and therapeutic implications of increased PGI₂ production by the arteries due to the dipyridamole-pentoxifylline combination remains to be ascertained.     

Increased platelet activity after termination of prostacyclin infusion into man

Infusion of PGI₂ at a dose of 5 or 10 ng/kg/min during 72 hours into patients with peripheral vascular disease was followed by increased susceptibility of platelets to proaggregatory action of ADP and collagen but not that of arachidonate. The above effects were observed 24 hours after termination of infusion of PGI₂. A tendency to an increased formation of TXA₂ in PRP aggregated by arachidonate was also noticed. Infusion of PGI₂ at a dose of 2 mg/kg/min during 72 hours into the patients caused the decreased platelt aggregability to ADP and arachidonate but not to collagen, and a decreased tendency of production of TXA₂ in PRP aggregated by arachidonate. The existence of a “rebound effect” in platelets after a long term PGI₂ therapy is suggested.     

The use of prostacyclin in the separation from plasma and washing of human platelets

A new method for the separation from plasma and washing of human platelets is described. The use of prostacyclin (PGI₂) throughout the procedure prevents the activation of platelets. The method allows a 60–70% yield of platelets from PRP. The platelet sensitivity to ADP, collagen, adrenaline, arachidonic acid and thrombin is the same as in PRP. The platelet suspension is stable for long periods and the reactivity to aggregating agents remain unchanged for periods greater than 48 h when platelets are stored at 4°C.     

Sex-related difference in the effect of aspirin on prostaglandin metabolism in rat platelet and aorta

The effect of aspirin on the production of the arterial prostacyclin (PGI₂)-like substance and platelet malondialdehyde (MDA) was investigated in rats of both sexes. No significant sex difference observed with the arterial PGI₂-like substance. But, following the aspirin treatment, the production of the PGI₂-like substance was significantly decreased in male rats. There was significant sex difference in the production of platelet MDA before the aspirin treatment. And after the aspirin treatment, platelets of both sexes produced significantly less MDA. It is possible that sex difference in the effect of aspirin is related to the quantitative difference of cyclooxygenase activity between platelets and vasal wall.     

Platelet-vessel wall interactions: Effects of platelets and plasma on the antiaggregatory activity and 6 keto-PGF1α production in isolated perfused aortas

An experimental model for the study of platelet-vessel wall interactions has been developed, based on perfusion of rat platelet-rich plasma (PRP) through isolated rat aortas. In the perfused PRP, platelet aggregation was inhibited and levels of 6 Keto PGF_1α and cAMP were elevated over the values found in non perfused PRP. When PPP or buffer were perfused through the isolated artery, elevations of 6 Keto PGF_1α levels in the perfusate were smaller (in perfused PPP) or of shorter duration (in both perfused PPP and buffer). The presence of platelets in the perfusion fluids thus enhanced the formation of Prostacyclin by the arterial wall. Levels of 6 Keto PGF_1α in PRP obtained from aspirin-treated animals and in PRP from normal animals, both perfused through normal aortas, were the same, and also levels of the above metabolite in normal PRP perfused through aortas of aspirin-treated animals did not differ from those found in non perfused PRP. It is concluded, from these data, that PRP does not stimulate PGI₂ formation in perfused aortas by providing cyclic endoperoxides. The experimental model developed allows the study of interactions between normal platelets and aortas from experimentally treated animals or viceversa.     

Stability of prostaglandin I2 in human blood

Prostaglandin I₂ (PGI₂) is hydrolyzed more slowly in human plasma than in buffer at the same pH; this stabilization appears to be due to binding of PGI₂ to albumin. The stability of the antiaggregatory potency of PGI₂ is similar in human blood and in plasma buffered to pH 7.55. No measurable conversion of [³H]-PGI₂ to radioactive or nonradioactive metabolites occurred during incubation with blood.     

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wishar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI₂-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to Λ¹⁷-6-keto-PGF_1α, a stable metabolite of PGI₃, could not be detected by an incubation study of ¹⁴C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI₂-like substance produced by rat aorta is most likely to be PGI₂. itself and not PGI₃.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Anti-platelet aggregating and disaggregating activities of 6,9-methano PGI2

Anti-platelet aggregating and disaggregating activities of the chemically stable 6,9-methano prostaglandin I₂ (6,9-methano PGI₂) were investigated. 6,9-Methano PGI₂ inhibited ADP-induced platelet aggregation in PRP from humans, rabbits and rats. 6,9-Methano PGI₂ also inhibited rabbit platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and 11,9-epoxy-methano PGH₂. Antiaggregating activities of 6,9-methano PGI₂ were 0.3 to 2.0 times greater than those of PGE₁. 6,9-Methano PGI₂ facilitated platelet disaggregation in a dose related manner. Antiaggregating and disaggregating activities of 6,9-methano PGI₂ were markedly enhanced by incubation with the phosphodiesterase inhibitor, theophylline.     

Generation of prostacyclin-like substance and lipid peroxidation in vitamin E-deficient rats

Endogenous generation of prostacyclin (PGI₂)-like substance and lipid peroxidation were studied in the aorta of rats fed on vitamin E-deficient diet and/or vitamin E-supplemented one for 4 to 10 months after they were weaned at 4 weeks. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and was estimated by comparison of its antiaggregatory activity with that produced by known amounts of synthetic PGI₂. Thiobarbituric acid-reacting substance (TBARS) was determined as an indicator of lipid peroxidation. The generation of PGI₂-like substance was significantly reduced in rats fed on vitamin E-deficient diet for 8 and 10 months as compared with that in the animals fed on vitamin E-supplemented one for the same period (p<0.001). Mean concentration of TBARS in the aortae of rats fed on vitamin E-deficient diet for 10 months was significantly higher than that of the animals fed on vitamin E-supplemented diet for the same feeding period (p<0.001). These alterations in the aortae of rats fed on vitamin E-deficient diet were corrected by feeding them on vitamin E-supplemented diet for subsequent 2 months.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI₂) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 μM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI₂ formation. The inhibition was not specific for PGI₂; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-¹⁴C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI₂ formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI₂.     

Prostacyclin (PGI2) effets on anterior pituitary hormones in the rat in vivo

Intravenous injection of 600 μg PGE₂ or PGI₂ significantly increased serum LH and prolactin levels in estradiol treated ovariectomized rats. There was no effect on serum FSH concentration. PGE₂ and PGI₂ stimulated LH release in a non-dose dependent manner, while prolactin levels were positively correlated with the dose administered following PGI₂ treatment. 6-keto-PGF_1α at a comparable dose had no effect on pituitary hormone levels. Subcutaneous administration of 1 mg/kg or 60 mg/kg PGI₂ for seven days significantly depressed serum LH level both in male and female rats. These doses had no effect on serum FSH or prolactin levels.     

Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts.

The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A₂, synthesized by platelets, and prostacyclin (PGI₂), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI₂ from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI₂ per 10⁵ cells, with a range of 0.2–5.3 ng PGI₂ per 10⁵ cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI₂ per 10⁵ cells with a range of 2.3–14.0 ng per 10⁵ cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI₂ per 10⁵ cells with a range of 0.05–0.6 ng per 10⁵ cells. When cultured cells were mixed with platelets, PGI₂ synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI₂ synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI₂ synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A₂ synthesis without compromising the capacity of the vasculature to produce PGI₂.     

Effect of prostacyclin on platelet-activating factor induced rabbit platelet aggregation

In this paper, the effect of prostacyclin (PGI₂) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI₂ and PGI₂-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of ³H-serotonin (³H-5HT).     

Mass-spectrometric evidence for spontaneous and angiotensin-induced generation of prostacyclin by perfused cat lungs

Isolated perfused cat lungs secreted spontaneously a PGI₂-like substance that relaxed a strip of bovine coronary artery. The presence of PGI₂ was confirmed by the identification of 6-oxo-PGF_α by GC-MS. Both bioassay and mass fragmentography showed that PGI₂ was released at a rate of 4 – 12 ng/ml. Generation of PGI₂ by the perfused cat lungs was stimulated by angiotensin II (3 – 10 ng/ml). The above results along with our $\text{in vivo}$ experiments point to the lung as an important source of endogenous PGI₂ in the body.