Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2 immediately prior to the initiation of irreversible aggregation. Selective thromboxane synthetase inhibitors block thromboxane A2 formation and aggregation. Thromboxane A2 formation appears to be essential during arachidonate mediated aggregation. The results presented reconcile the previously accepted paradoxical behavior of thromboxane synthetase in platelet rich plasma toward the prostaglandin endoperoxide H2 substrate.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid Two selective thromboxane synthetase inhibitors

Two selective thromboxane A₂ synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B₂ and prostaglandin E₂ accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B₂ formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H₂. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A₂ formation with an accompanying, parallel, suppression of the platelet aggregation.Imidazole selectively suppressed thromboxane A₂ formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H₂ on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A₂ formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A₂ in arachidonate mediated platelet aggregation.     

Synthesis of thromboxane A2 by non-aggregating dog platelets challenged with arachidonic acid or with prostaglandin H2

Dog platelets challenged with arachidonic acid fail to aggregate but synthesize a substance which aggregates rabbit and human platelets, this aggregation being suppressed by dibutyryl cyclic AMP. The aggregating substance contracts strips of rabbit aorta and of coeliac and mesenteric arteries, is soluble in diethyl ether, has a half-life of about 40 seconds at 37°C and of 100 seconds at 22°C. Its generation is blocked by various inhibitors of prostaglandin biosynthesis. The thromboxane A₂ synthetase inhibitor imidazole and its analogue benzimidazolamine also suppress generation of vessel contracting activity in incubates of dog platelets and prostaglandin H₂. Since dog platelets also transform prostaglandin H₂ into thromboxane A₂ their failure to aggregate, when stimulated by arachidonic acid or by prostaglandin H₂, is not due to lack of thromboxane synthesizing ability.     

Inhibition of PGE1-stimulated cAMP accumulation in human platelets by thromboxane A2

The prostaglandin endoperoxide PGH₂, HHT, HETE, thromboxane A₂, and thromboxane B₂, which are all products of arachidonic acid metabolites of human platelets, were tested for their ability to modulate platelet cyclic nucleotide levels. None of the compounds tested altered the basal level of cAMP or cGMP, and only PGH₂ and thromboxane A₂ inhibited PGE₁-stimulated cAMP accumulation. Thromboxane A₂ was found to be a more potent inhibitor of PGE₁-stimulated cAMP accumulation and inducer of platelet aggregation thatn PHG₂.     

A selective thromboxane synthetase inhibitor blocks the cAMP lowering activity of PGH2.

The thromboxane synthetase inhibitor, 9,11-azoprosta-5,13-dienoic acid, blocks both platelet aggregation and the cyclic AMP lowering activity of the prostaglandin endoperoxide PGH₂. These data indicate PGH₂ must be converted into thromboxane A₂ in order to lower cAMP or induce platelet aggregation.     

Kinetic studies on the conversion of prostaglandin endoperoxide PGH2 by thromboxane synthase

We have investigated the time course of formation of thromboxane A₂, thromboxane B₂, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A₂, but rather thromboxane A₂ decomposes exclusively into thromboxane B₂. The kinetics of formation of thromboxane B₂ from the endoperoxide prostaglandin H₂ in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A₂ from prostaglandin H₂ and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H₂.     

A method for measuring the unstable thromboxane A2: Radioimmunoassay of the derived mono-o-methyl-thromboxane B2

A radioimmunoassay was developed for a mono-O-methyl derivative of thromboxane B₂. The antibodies showed high specificity for this compound and cross reacted only 1.2% with thromboxane B₂ and less than 0.1% with prostaglandins and prostaglandin metabolites. The method had a sensitivity of 7 picog. The radioimmunoassay was employed in studies where thromboxane A₂ was generated in human platelets and immediately converted into mono-O-methyl thromboxane B₂ by treatment of the sample with a large volume of methanol. In some of the experiments, thromboxane B₂ was simultaneously measured by a separate radioimmunoassay. Using these two assays it was demonstrated that thromboxane A₂ could be detected only during the earlier stages of the platelet aggregation, whereas thromboxane B₂ rapidly reached a constant level. In a separate experiment, the half-life of thromboxane A₂ in buffer was found to be 32.5±2.5 (S.D.) sec at 37°C; the compound was more stable at lower temperatures. The for thromboxane A₂ was also considerably longer in plasma.     

Characterization of imidazo[1,5-a]pyridine-5-hexanoic acid (CGS 13080) as a selective thromboxane synthetase inhibitor using in vitro and in vivo biochemical models

CGS 13080 inhibited cell-free thromboxane synthetase with an IC₅₀ of 3 nM. It was at least five orders of magnitude less potent toward other key enzymes involved in arachidonic acid metabolism. Submicromolar concentrations inhibited calcium ionophore-induced formation of thromboxane B₂ by intact human platelets with concomitant accumulation of prostaglandin E₂. Oral doses lower than 1 mg/kg in rats suppressed the elevations of plasma thromboxane B₂ induced by calcium ionophore. This was attended by shunting of endoperoxide substrate to 6-keto-prostaglandin F₁α and prostaglandin E₂. CGS 13080 is one of the most potent and selective thromboxane synthetase inhibitors yet identified.     

Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

Thromboxane A₂ plays and important role in arachidonic acid- and prostaglandin H₂-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I₂, prostaglandin I₁, and prostaglandin E₁) and dibutyryl cyclic AMP inhibit both thromboxane A₂ formation and arachidonate-induced aggregation platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A₂ is still formed. Prostaglandin H₂-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A₂ production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cycyooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate mode for studying relationship between the platelet cyclooxygenase and platelet function.     

Thromboxane A2: Effects of airway and vascular smooth muscle

Thromboxane A₂, an unstable compound derived from prostaglandin G₂, weas generated by incubation of arachidonic acid with a suspension of human platelets. The activity of thromboxane A₂ relative to that of prostaglandin H₂ in causing contractions of a number of smooth muscle organs were as follows: rabbit aorta, 7–20; human umbilical artery, 9–60; and guinea pig trachea, 2–12. Intravenous injection of thromboxane A₂ into anaesthetized guniea pigs was followed by a pronouced increase in the tracheal insufflation pressure, potency compared to prostaglandin H₂, 31–45.     

Thromboxane A2 and prostaglandin H2: Potent stimulators of the swine coronary artery

Thromboxane A₂ was generated by incubation of arachidonic acid with a suspension of human platelets. The filtrate contained 266 ± 46 ng/ml (n=10) of thromboxane A₂ and 25 ng/ml or less of prostaglandin endoperoxides (prostaglandins G₂+H₂). Thromboxane A₂ was 2–10 times more potent than prostaglandin H₂ and 9–102 times and 26–308 times more potent than prostaglandins E₂ and F₂α, respectively, in causing contractions of the superfused swine coronary artery.     

Polymorphonuclear leukocytes produce thromboxane A2-like activity during phagocytosis

Homogenates of phagocytosing polymorphonuclear leukocytes obtained from rabbit peritoneum were incubated with the prostaglandin endoperoxides PGG₂ or PGH₂. After 2 min at 0°C, incubation mixtures contained an increased rabbit aorta contracting activity. Ether extracts of incubation mixtures contained a substance which contracted the superfused strips of rabbit aorta and coeliac artery and had a half life which was similar to thromboxane A₂. The generation of thromboxane A₂-like activity from PG endoperoxides was prevented by boiling the homogenate prior to incubation, or by pretreatment with benzydamine, a drug which blocks thromboxane formation in platelets. Production of thromboxane A₂-like material by leukocyte homogenates was compared with platelet microsomal thromboxane synthetase.     

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A₂ biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10^?4 M, 1.75 × 10^?4 M, and 2 × 10^?3 M respectively. Their relative inhibitory potencies on thromboxane B₂ production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10^?3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B₂ formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A₂ biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.     

Enzymatic conversion of C21 endoperoxides to thromboxanes and hyroxy acids

ω-Homo and α-homo [³H₈]prostaglandin H₂ were prepared by chemical and enzymatic methods from 5,8,11,14-heneicosatetraynoic acid and 6,9,12,15-heneicosatetraynoic acid. Upon incubation with human platelet microsomes, two products were formed from each endoperoxide, viz. ω-homo thromboxane B₂ plus 12-hydroxy-octadecatrienoic acid and α-homo thromboxane B₂ plus 13-hydroxy-octadecatrienoic acid. In the light of previous results this suggests that the distance between the double bond of the carboxyl side chain and the cyclopentane ring of prostaglandin H₂ may be important for conversion to thromboxanes.     

Biosynthesis of prostacyclin (PGI2) and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) by pericardium, pleura, peritoneum and aorta of the rabbit

Prostacyclin generation by pericardium, pleura, peritoneum, aorta and dura mater of the rabbit was assessed as platelet aggregation inhibitory activity in platelet rich plasma. All tissues except the dura mater, were also incubated with labelled (1-¹⁴C) arachidonic acid and (1-¹⁴C) prostaglandin endoperoxide H₂ and the various metabolites formed were identified radiochromatographically. Pericardium, pleura and peritoneum form substantially high amounts of prostacyclin and HETE indicating that these tissues contain both cyclo-oxygenase and prostacyclin-synthetase. They also show considerable lipoxygenase activity.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A₂, thromboxane A₂-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholinase A₂. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A₂-like activity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A₂.The results indicate that formation of thromboxane A₂-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: Pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to supress aggregation, through the action of their respective non-enzymatic breakdown products PGE₁, PGD₂, or PGD₃ all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cycloogygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A₂, and enhance formation of the anti-aggregatory metabolite, prostacyclin.Whereas EPA is not detectably metabolized by platelets, dihomo-γ-linolenic acid (8,11,14,-eicosatrienoic acid) is primariley converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH₁ and DLL which was partially compromised by the PGE₁ formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH₁. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A₂.     

Biosynthesis of prostaglandin D2 1. Formation of prostaglandin D2 by human platelets

Formation of prostaglandin D₂ (PGD₂) during the aggregation of platelets was determined, employing a specific bioassay. PGD₂ was synthesized in human platelet rich plasma (PRP) in response to thrombin, collagen and epinephrine. Indomethacin pretreatment abolished the biosynthesis of PGD₂. When thrombin treated PRP was incubated for different periods of time and denatured in the presence of SnCl₂ to prevent the formation of PGD₂ from endoperoxides during the extraction procedure, PGD₂ formation was noted within the first minute of incubation and reached a peak level after 4 minutes. PGD₂ from thrombin stimulated PRP was conclusively identified by gas chromatography-mass spectrometry.The formation of PGD₂ during platelet aggregation could represent a mechanism of feedback inhibition of aggregation.     

Interralation of prostaglandin endoperoxide (prostaglandin G2) and cyclic 3′,5′-adenosine monophosphate in human blood platelets

The prostaglandin endoperoxide, prostaglandin G₂, in platelet-rich plasma may produce reversible platelet aggregation without secretion, irreversible aggregation with secretion of platelet constituents inhibited by indomethacin, or the latter effects despite indomethacin, depending on the concentration of the endoperoxide. Irreversible aggregation and platelet secretion induced by prostaglandin G₂ apparently result from the action of ADP, since these responses are inhibited by 2-n-amylthio-5′-AMP (an inhibitor of the actions of ADP on platelets) and they do not occur in heparinized platelet-rich plasma. Prostaglandin G₂ lowers the platelet level of cyclic 3′,5′-AMP. Its actions are inhibited by elevation of cyclic AMP levels by prostaglandin E₁ or dibutyryl cyclic AMP or adenosine. Like malondialdehyde production induced by thrombin, ADP, or arachidonic acid, prostaglandin G₂-induced malondialdehyde production is reduced by dibutyryl cyclic AMP and prosraglandin E₁. Platelet activation by prostaglandin G₂ is enhanced by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)-adenine.The action of prostaglandin G₂ on platelets is more complex then previously reported.     

PGH2, TxA2 and PGI2 have potent and differentiated actions on human uterine contractility

The contractile response to three different prostanoids of the isolated human myometrium and the different layers of the utero-tubal junction (UTJ) was studied $\text{in vitro}$. The prostaglandin endoperoxide, PGH₂, stimulated contractility of both the myometrium and the outer and inner muscle layers of the UTJ, whereas the intermediate layer of the UTJ was inhibited. Thromboxane A₂ generated from PGH₂ and a thromboxane synthase preparation caused a stimulation of both the myometrium and all three layers of the UTJ. The stimulatory response to TxA₂ occurred at concentrations as low as 50–70 pg/ml. The sodium salt of PGI₂ was found to relax both the myometrium and all the layers of the UTJ. Intravenous administration of PGI₂ in repeated doses between 2–8 μg induced facial flushing and headache but had little if any effect on $\text{in vivo}$ uterine contractility. At least under $\text{in vitro}$ conditions, these short-lived prostanoids and/or their metabolites apparently have a specific action on uterine contractility, an action which is manifested at comparatively low concentrations.