[The role of prostaglandins in platelet aggregation in metabolic syndrome].

The role of metabolic products of arachidonic acid and thromboxans in metabolic syndrome was evaluated in 42 patients and 16 healthy subjects. The levels of arachidonic acid and thromboxane were shown to be elevated in patients with metabolic syndrome which accounted for enhanced platelet aggregation in response to ADP, adrenaline, and collagen. It is concluded that that decreased level of cyclic nucleotides (cAMP) and prostacyclin in combination with a rise in the content of Willebrand factor in patients with metabolic syndrome is a major contributor to the development of platelet activity.

Photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor.

The synthesis, binding and photoincorporation of a thromboxane A2/prostaglandin H2 (TXA2/PGH2) analog (9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-azidophenyl )-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2) [( 125I]PTA-Azido) to washed human platelets was characterized. Kinetic analysis of the binding of [125I]PTA-Azido at 30 degrees C yielded a k1 of 1.83.10(7) M-1.min-1 and k -1 of 0.195 min-1, Kd = k -1/k1 = 11 nM. Incubation of washed human platelets with [125I]PTA-Azido followed by photolysis resulted in the radiolabelling of a number of platelet proteins as assessed by SDS-PAGE autoradiography. The radiolabelling of three of these protein bands could be either uniformly blocked or reduced with a series of structurally dissimilar TXA2/PGH2 receptor antagonists or agonists and corresponded to proteins with a molecular mass of 43, 39 and 27 kDa. In addition, the incorporation of [125I]PTA-Azido into the three proteins was stereoselectively blocked by a pair of optically active stereoisomers that are TXA2/PGH2 receptor antagonists. Two-dimensional gel electrophoresis indicated that the 43 kDa protein possessed a pI value of 5.6 and that the 27 kDa protein exists in at least three isoforms with pI values of 4.9, 5.1 and 5.3. The labelling pattern was not altered by a mixture of proteinase inhibitors. The data suggest that one or more of these specifically radiolabelled proteins may represent the human platelet TXA2/PGH2 receptor.

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor.

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.

Prostaglandin endoperoxides, thromboxane A2 and adenosine diphosphate in collagen-induced aggregation of rabbit platelets.

1 A bioassay technique is described for simultaneously monitoring rabbit platelet aggregation with measurement of thromboxane A(2) (TxA(2)) and prostaglandins released in response to collagen or arachidonic acid (AA).2 Five imidazole derivatives were examined as inhibitors of thromboxane synthetase and compared with the effect of the cyclo-oxygenase inhibitor indomethacin; 1-(7-carboxyheptyl) imidazole was identified as the most potent and selective inhibitor of thromboxane synthetase and was used with indomethacin to investigate the relative contribution of the prostaglandin endoperoxides prostaglandin G(2) (PGG(2))/PGH(2) and TxA(2) in mediating platelet aggregation induced by collagen or AA.3 Platelet aggregation induced by a low concentration of collagen was abolished by indomethacin and carboxyheptylimidazole whilst in response to a high concentration or collagen only partial inhibition of aggregation occurred.4 The contribution of adenosine diphosphate (ADP) released from platelets during collagen or AA-induced aggregation was examined using the substrate/enzyme complex creatine phosphate/creatine phosphokinase (CP/CPK). The CP/CPK complex abolished aggregation induced by a low dose of collagen whilst aggregation to a high dose of collagen was only partially inhibited.5 Aggregation induced by a high dose of collagen was abolished by a combination of CP/CPK with indomethacin or carboxyheptylimidazole.6 AA-induced aggregation was abolished by indomethacin. Carboxyheptylimidazole abolished aggregation induced by a low dose of AA but inhibition was surmounted with increasing concentrations of AA in the absence of TxA(2) formation.7 PGH(2)-induced aggregation was unaffected by indomethacin and only partially inhibited by carboxyheptylimidazole. AA or PGH(2)-induced platelet aggregation was unaffected by CP/CPK.8 In conclusion, aggregation of rabbit platelets induced by a low concentration of collagen was dependent on synergism between TxA(2) and ADP whilst at high concentrations of collagen, sufficient TxA(2) and ADP were released to induce aggregation independently of each other.9 The small amounts of prostaglandin endoperoxides produced from endogenous arachidonate have apparently no direct pro-aggregatory role. However, the relatively large amount which can be produced by a high concentration of exogenous AA when TxA(2) formation is prevented can cause aggregation of rabbit platelets.

Effect of thromboxane A2 synthetase inhibition, singly and combined with thromboxane A2/prostaglandin endoperoxide receptor antagonism, on inositol phospholipid turnover and on 5-HT release by washed human platelets.

Differential effects on human platelet function of thromboxane A2 (TXA2) synthetase inhibition singly and of TXA2 synthetase inhibition combined with TXA2/prostaglandin endoperoxide receptor antagonism were revealed, using ridogrel as a probe. Ridogrel combines selective TXA2 synthetase inhibition with TXA2/prostaglandin receptor antagonism in one molecule: in washed human platelets, the compound reduces the production of TXB2 (IC50 = 1.3 X 10(-8) M) and increases that of PGF2 alpha, PGE2, PGD2 from [14C]arachidonic acid. Additionally, at higher concentrations (Ki = 0.52 X 10(-6) M), it selectively antagonizes the breakdown of inositol phospholipids, subsequent to stimulation of TXA2/prostaglandin endoperoxide receptors with U 46619. The latter happens in a competitive way with fast receptor association-dissociation characteristics. At low concentrations (1 X 10(-9)-1 X 10(-7) M) producing single TXA2 synthetase inhibition, ridogrel reduces the collagen-induced formation of TXB2 by washed platelets, but enhances [32P]phosphatidic acid (PA) accumulation and [3H]5-hydroxytryptamine (5-HT) release. At higher concentrations (1 X 10(-6)-1 X 10(-5) M) which additionally block U 46619-induced [32P]PA accumulation, ridogrel inhibits the [32P]PA accumulation and release of [3H]5-HT by human platelets stimulated with collagen. These observations, corroborated by results obtained with OKY 1581, sulotroban, indomethacin and human serum albumin, suggest a causal role for prostaglandin endoperoxides in the stimulation by TXA2 synthetase inhibition of platelet reactions to collagen. They reinforce the concept that TXA2 synthetase inhibition-induced reorientation of cyclic endoperoxide metabolism, away from TXA2 into inhibitory prostanoids, requires additional TXA2/prostaglandin endoperoxide receptor antagonism to achieve optimal anti-platelet effects.

Characterization of U46619 binding in unactivated, intact human platelets and determination of binding site affinities of four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548).

The binding of U46619 and the inhibition of this binding by four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548) were studied in unactivated, intact human platelets. Washed platelets were equilibrated with [3H]-U46619 (5 nM) and the time course of binding determined. The receptor-specific binding reached equilibrium within 2-4 minutes, and could be displaced by addition of excess unlabelled ligand. Saturation of this binding was achieved at 750 nM. Scatchard transformation of the saturation binding curve yielded a single class of binding site with a Kd of 108 nM and Bmax of 360 fmole/10(8) platelets. When [3H]-U46619 (4 nM) was incubated with platelets in the presence of increasing concentrations of the antagonists, binding of U46619 was inhibited in a dose dependent manner. The potency series for inhibition of U46619 binding was: SQ 29,548 (IC50 = 7.9 nM) greater than ONO 3708 (IC50 = 38 nM) greater than BM 13.177 (IC50 = 0.91 microM) greater than 13-APA (IC50 = 6.2 microM). These findings are consistent with the notion that these compounds all act as competitive antagonists at the level of the platelet TXA2/PGH2 receptor.

A thromboxane synthetase inhibitor reorients endoperoxide metabolism in whole blood towards prostacyclin and prostaglandin E2.

During incubation of citrated blood at 37 degrees C the levels of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and prostaglandin E2 (PGE2) remain constant, but rise markedly within one minute after the addition of collagen, particularly when thromboxane synthetase is blocked. The amount of 6-keto PGF1 alpha formed is dose-dependent for both collagen and the thromboxane synthetase inhibitor (UK-37,248). Moreover, the number of platelets will determine the extent of the 6-keto PGF1 alpha jump, that does not occur when blood is drawn after aspirin ingestion. The production of 6-keto PGF1 alpha in function of time is composed of a fast platelet-related (intercept) and a slower probably leukocyte-dependent contribution (slope). In the absence of UK-37,248 the intercept is 115 +/- 85 pg/ml, the slope is 12.9 +/- 7.7 pg/min/ml whereas in the presence of the thromboxane synthetase inhibitor they are 411 +/- 177 pg/ml and 56.2 +/- 25 pg/min/ml respectively. The present findings indicate that a thromboxane synthetase inhibitor, by not only reducing thromboxane A2 production but also enhancing prostacyclin generation with blood is exposed to thrombogenic stimuli such as collagen, should be superior to aspirin as an antithrombotic agent, although possible interference by enhanced PGE2 production should be taken into account.

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 alpha. UK 37,248 inhibited TXB2 formation by greater than 95% and increased the production of PGD2, PGE2 and PGF2 alpha 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.

Contribution of arachidonic acid metabolites to atherosclerosis.

Evidence is presented that arachidonic acid metabolites are involved to an important extent in platelet-arterial wall interaction. The perspectives opened up by these results are the possible prevention and therapy of atherosclerosis by a fish-diet containing eicosapentaenoic acid, the use of prostaglandins, releasers of endogenous PGI2 and thromboxane synthetase inhibitors.

Soluble plasma fibrin and platelet prostaglandin endoperoxides following myocardial infarction.

Following myocardial infarction, soluble fibrin in plasma is often elevated as a sign of an activated plasmatic coagulation system. Soluble fibrin in plasma is most pronounced in shock patients under catecholamine administration. With the improvement of the clinical situation the fibrin concentration declines to normal. Following incubation with N-ethylmaleimide an increased production of prostaglandin endoperoxides is observed in the platelets of patients after myocardial infarction compared to normal platelets. Plasma of patients exhibits an endoperoxide-producing effect on normal platelets. The stimulating effect of patient plasma diminishes together with the fall of soluble plasma fibrin. These phenomena may be considered as signs of a relation between the plasmatic and thrombocytic coagulation system.

Interactions of prostaglandin H2 and thromboxane A2 with human serum albumin.

The present report describes the interactions of human plasma proteins with the unstable endoperoxide, prostaglandin H2 and thromboxane A2, generated by incubation of platelets with prostaglandin H2 or arachidonic acid. It was found that both compounds reacted very rapidly with plasma proteins to form covalently bound derivatives. The major reacting plasma protein was human serum albumin. Depending on conditions, 20-40% of added prostaglandin H2 and 50-80% of generated thromboxane were bound to proteins. This reaction of both prostaglandin H2 and thromboxane A2 prevents their detection by classical analytical methods. The protein binding of thromboxane was more pH-sensitive than the binding of prostaglandin H2. The reactions cause reduced levels of both endoperoxide and thromboxane B2 in suspensions of washed platelets using human serum albumin as compared to buffer. It was also shown that the half-life of prostaglandin H2 was considerably reduced in the presence of albumin.

Platelet dysfunction and alteration of prostaglandin metabolism after chronic alcohol consumption.

To study the effects of chronic alcohol consumption on platelet functions, the rate of arachidonate-induced platelet aggregation, the production of malondialdehyde in platelets, and plasma levels of prostaglandin endoperoxide metabolites were examined in 88 chronic alcoholics and 24 healthy controls. The rate of platelet aggregation and the production of malondialdehyde in platelets were greater in chronic alcoholics both on admission and 1 week after. However, these alterations returned to the level of healthy controls within 4 weeks of abstinence from alcohol and were independent of the number of circulating platelets. Furthermore, on admission, plasma levels of thromboxane B2 were significantly increased in chronic alcoholics when compared with those of healthy controls (400.8 +/- 36.5 versus 241.7 +/- 28.9 pg/ml plasma; p less than 0.025) and were also significantly correlated with malondialdehyde production in washed platelet debris (r = 0.6049; p less than 0.001). In contrast, plasma levels of 6-keto prostaglandin F1 alpha and prostaglandin E were not altered after chronic alcohol consumption. As a result, the ratio of 6-keto prostaglandin F1 alpha to thromboxane B2 was markedly decreased in chronic alcoholics (0.31 +/- 0.03 versus 0.62 +/- 0.13; p less than 0.001). These results strongly suggest that the imbalance in prostaglandin endoperoxide metabolites is produced by chronic alcohol ingestion. Moreover, a significant correlation was observed between platelet aggregation rate and malondialdehyde production during platelet aggregation (r = 0.559; p less than 0.005). Thus, we conclude that chronic alcohol consumption alters platelet thromboxane metabolism, which is likely associated with the increased ability of platelets to aggregate.

[Kinetic mechanisms of enzyme activity of the thromboxane synthetase system. Thromboxane synthetase of human platelets]. / Kineticheskie zakonomernosti deistviia fermentov tromboksansinteziruiushchei sistemy. Tromboksansintetaza trombotsitov cheloveka.

Partially purified preparations of prostaglandin endoperoxide-synthetase (PGH-synthetase) and thromboxane synthetase (PGH-convertase) were obtained from human platelets by ion exchange chromatography. The kinetics of prostaglandin H2 enzymatic conversion was studied in the presence of thromboxane synthetase from human platelets. It was found that prostaglandin H2 conversion into both thromboxane A2 and malonic dialdehyde is a catalytic process, i. e., its rate increases as the protein concentration rises. The linear dependence of the enzymatic reaction velocity on substrate concentration at a prostaglandin H2 concentrations below 10-15 microM was demonstrated. PGH-synthetase and PGH-convertase from human platelets exhibit similar enzymatic activity dependence on pH and temperature, PGH-convertase being more thermostable.