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WATANABE, J., UMEDA, F., WAKASUGI, H. and IBAYASHI, H. Effect of Vitamin E on Platelet Aggregation in Diabetes Mellitus. Tohoku J. exp. Med., 1984, 143(2), 161-169 - Vitamin E is known to be an inhibitor of platelet prostaglandin production and aggregation. The rate of platelet aggregation induced by adenosine diphosphate was significantly increased in diabetics with proliferative retinopathy, and the enhanced production of thromboxane B2, a stable metabolite of thromboxane A2, was demonstrated in those patients. On the other hand, vitamin E was significantly reduced in platelets obtained from diabetics as compared to age matched control subjects. In addition, it was shown that vitamin E content in platelets examined in diabetic and control subjects inversely correlated with both the rate of platelet aggregation and thromboxane B2 production during aggregation. It is suggested that the reduced vitamin E levels in diabetic platelets can contribute to the mechanisms of the enhanced platelet thromboxane A2 production and aggregation which lead to the development of vascular complications.

Endotoxin (0.2-20 μg/ml) induced a limited, dose-dependent change in light transmission when incubated with canine platelet-rich plasma. This response differed from that induced by collagen and ADP in its magnitude (ca. 25%) and by its insensitivity to a wide variety of inhibitors. However, endotoxin (20-1000 μg/ml) did not induce detectable aggregation of canine platelets in citrated whole blood under conditions where collagen and ADP were effective. In addition, thromboxane production was not stimulated. These data are consistent with the occurrence of an immune adherence reaction between endotoxin and canine platelets, as has been previously described for other species, but without platelet activation (aggregation, mediator, and thromboxane release).

The production of prostacyclin (PGI2) and thromboxane (TXA2) by irradiated rat lung and the concentration of these prostanoids in the bronchoalveolar lavage (BAL) fluid were measured in rats sacrificed at 2 or 6 months after a single dose of 10-30 Gy of cobalt-60 gamma rays to the right hemithorax. Lung PGI2 and TXA2 production increased linearly with increasing radiation dose at rates of 3.1% and 4.3% per Gy, respectively, at 2 months and 21.7% and 6.1% per Gy, respectively, at 6 months. At both times, PGI2 and TXA2 production tended to plateau at doses of 25 Gy or higher. This plateau usually disappeared in the presence of exogenous arachidonic acid, suggesting that the Vmax for PGI2 production at 2 and 6 months and for TXA2 at 6 months was substrate-limited rather than enzyme-limited. At 2 months after irradiation, the modifying agents D-penicillamine (an inhibitor of collagen cross-linking) and Captopril (an angiotensin-converting enzyme inhibitor) exhibited significant dose-reduction factors of 1.3-1.8 for PGI2 production and 1.3-1.4 for TXA2 production. In the shielded left lung, PGI2 production increased slightly but significantly with increasing contralateral dose at 2 months postirradiation. The concentration of TXA2 in the BAL fluid increased linearly with increasing radiation dose at 2 months after exposure, whereas PGI2 concentration was independent of dose. At 6 months, the reverse was true. These data demonstrate that hemithorax irradiation produces a dose-dependent increase in pulmonary PGI2 and TXA2 production for at least 6 months, and suggest that BAL data may serve as an index of radiation-induced pulmonary damage.

Generation of reactive oxygen metabolites, thromboxane increases, and vasoconstriction have been implicated in the pathogenesis of acute edematous lung injury, such as that seen in patients with the Adult Respiratory Distress Syndrome (ARDS), but their interactions are unknown. We hypothesized that reactive O2 products would stimulate arachidonic acid metabolism in lungs and that vasoactive products of arachidonate, such as the potent vasoconstrictor thromboxane A2, might then mediate O2-metabolite-induced pulmonary vasoconstriction. We found that O2 metabolites generated by injection of purine plus xanthine oxidase caused increases in mean pulmonary artery perfusion pressures (27 +/- 4 mmHg) in isolated perfused lungs. In addition, purine plus xanthine oxidase also caused 30-fold increases in perfusate levels of thromboxane B2 (the stable metabolite of thromboxane A2) compared with only twofold increases in 6-keto-PGF1a (the stable metabolite of prostacyclin). Moreover, prior addition of catalase inhibited both vasoconstriction and the thromboxane B2 production seen in isolated lungs following injection of purine plus xanthine oxidase. Similarly, pretreatment with cyclooxygenase inhibitors, either aspirin or indomethacin, also completely blocked thromboxane generation and markedly attenuated pressor responses usually seen after purine plus xanthine oxidase (increase in mean pulmonary artery perfusion pressures, 4.4 +/- 1.5 mmHg). Furthermore, imidazole, a thromboxane synthetase inhibitor, also decreased O2-metabolite-induced thromboxane generation and vasoconstriction. These results suggested that thromboxane generation might participate in O2-metabolite-induced vasoconstriction. However, since a significant correlation between thromboxane levels and the degree of vasoconstriction could not be demonstrated, and since addition of superoxide dismutase reduced thromboxane generation but did not affect the intensity of vasoconstriction, it is possible that thromboxane is not the only vasoactive mediator in this model. We conclude that exposing lungs to O2 metabolites results in thromboxane generation and that thromboxane is a major mediator of oxidant-induced vasoconstriction.

Mercuric chloride [25–50 μg/ml platelet-rich plasma (PRP)] lowers the threshold concentration of arachidonic acid (AA) required for triggering rabbit platelet aggregation and causes a marked increase of thromboxane production. The metal, added as HgCl2, does not modify (50.μg/m1 PRP) or block (100 μg/ml PRP) the platelet aggregation wave induced by a normal aggregating dose of AA. Whether or not AA-induced platelet aggregation takes place, a large increase in thromboxane production is observed. Methyl mercury, assayed as reference drug, induces platelet aggregation and a significant increase of thromboxane levels. Finally, HgC12 and methyl mercury, in a concentration range of 0.125–0.5.μg/μl in the incubation liquid, induce an increased prostacyclin release from rat aortic tissue.

The major arachidonic acid metabolites excreted by untreated rat heart endothelial cells were prostacyclin (PGI 2 ), 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Trace amounts of both prostaglandin E (PGE) and thromboxane B 2 (TXB 2 ) were also produced. The activity of leukotriene B 4 (LTB 4 ) was the same as baseline activity. Gamma-interferon (γ-IFN) treatment increased the production of PGI 2 and PGE more than three-fold compared to control values. Thromboxane production increased two-fold while the excretion of 5-HETE and 15-HETE was unaffected. No LTB 4 excretion was seen after γ-IFN stimulus. Methylprednisolone downregulated the 5-HETE, 15-HETE and prostacyclin production both in normal and γ-IFN treated cells. Indomethacin decreased prostacyclin excretion in untreated cells and also decreased γ-IFN-induced prostacyclin and PGE excretion. Taken together these results indicate that rat microvascular endothelial cells excrete prostaglandins and prostacyclin, but not leukotrienes.

A radioimmunoassay was developed for 11-dehydro-TXB 2 , a prominent metabolite of TXB 2 in blood and urine of several species. In order to reliably assay 11-dehydro-TXB 2 , its chemical stability as well as its chromatographic properties were first examined. Since dehydrogenation at C-11 converts the thromboxane ring into the δ-lactone form of a dicarboxylic acid, which can also occur in an open form, the analysis of 11-dehydro-TXB 2 may be somewhat complicated. In some chromatographic systems, the compound thus migrated with pronounced tailing, and during extraction using the common Sep-Pak procedure the two forms were partially separated. The lactone as well as the open form could be conclusively identified using mass spectrometry. The equilibrium between the two forms of 11-dehydro-TXB 2 was studied in buffers of different pH and in plasma. Higher pH favoured hydrolysis into the acyclic structure. The lactonization and hydrolysis processes were also shown to be time and temperature dependent. Two different antiplasmas, raised in rabbits against conjugates of 11-dehydro-TXB 2 with bovine serum albumin, displayed somewhat different properties in their recognition of the two forms of 11-dehydro-TXB 2 . A radioimmunoassay employing these antibodies was developed. The labeled antigen was prepared by incubation of H-TXB 2 with rabbit supernatant. The limit of detection was 1.5 pg. For validation of the assay, analysis of blood and urinary samples, obtained after injection of TXB 2 to a human volunteer, was done. The values obtained were compatible with previous isotope studies. Results from an inhibition experiment with rabbit lung incubated in the presence or absence of indomethacin further supported the identity of the assayed substance.

SummaryVascular prostacyclin (PGI2) production is different in the arteries and veins of the dog. Experiments were performed to determine whether chronic grafting of the femoral vein into the arterial circulation would alter the normal PGI2 and thromboxane (TxA2) synthesis of the “arterialized” veins. Spontaneous and arachidonic acid (AA) stimulated PGI2 and TxA2 production (measured by radioimmunoassay of 6-keto PGF1α and TxB2 respectively) were analysed in full thickness punch biopsies of the middle part of the grafts after 3 and 16 months and compared with unoperated veins and arteries. PGI2 production was significantly higher in arteries than in veins but no significant difference in TxB2 production was found. Middle “arterialized” venous graft produced significantly lower amounts of PGI2 and higher amounts of TxB2 than unoperated vessels. PGI2 production was more reduced in the distal than in the middle or the proximal parts of the venous grafts especially when stimulated with AA. These findings do not support the concept that the venous graft was biochemically adapted or “arterialized” in terms of PGI2 production when implanted for 3 months or longer. Rather the markedly decreased PGI2/TxB2 ratio in the middle of the graft may be a contributory cause of thrombogenicity and may be implicated in the pathogenesis of neointimal hyperplasia.

The circulation of lymph is apparently controlled partly by the pressure of fluid entering the lymphatic capillaries, aided by external forces such as skeletal muscle contraction and gut peristalsis1,2, but lymphatic vessels themselves also contract rhythmically when distended in vivo or in vitro and this may be of major importance in the propulsion of lymph3–8. We have studied the effects of biochemical mediators on isolated sheep and bovine lymphatic segments and on pressure pulses from indwelling lymphatic cannulae and report here that nanomolar concentrations of prostaglandin (PG) H2 endoperoxide and a stable PGH2 analogue elicited rhythmical contractions and increased the tone of isolated quiescent lymphatic segments. Some segments showed spontaneous rhythmic activity, and these spontaneous contractions could be inhibited not only by aspirin (a cyclo-oxygenase inhibitor) but also by inhibitors of thromboxane synthase (imidazole and compound UK 37248). The pulsatile pressure changes measured from indwelling lymphatic cannulae in conscious sheep were also suppressed by aspirin or imidazole infused directly into the lymphatic circulation. Our results suggest that endogenous thromboxane production is mainly responsible for spontaneous lymphatic contractions but that PGH2 also has contractile activity. An unknown product of arachidonate and PGH2, which could be formed non-enzymatically, inhibited both spontaneous and induced contractions.

Arachidonate-induced ex vivo platelet aggregations were performed before and after subjects received various doses of either ibuprofen or flurbiprofen. Both ibuprofen and flurbiprofen reduced the initial rate of thromboxane B2 (TxB2) production, but about 10% of control TxB2 production was refractory to either drug. Aggregation was observed after TxB2 concentrations exceeded a threshold of about 40 ng/ml. Once the threshold was achieved, aggregation and TxB2 production occurred independently of either drug. Ibuprofen and flurbiprofen concentrations of about 3 and 0.2 micrograms/ml, respectively, inhibited aggregation during the 5-minute testing period. A pharmacodynamic model was developed that predicts TxB2 production, aggregation lag times, and percent aggregation as a function of the time course of drug concentrations in serum.

The vascular wall has the capacity to produce thromboxane A2. However, the role of vascular thromboxane A2 is still uncertain. In this study, we examined the relationship between vascular thromboxane A2 generation and vascular smooth muscle cell growth in spontaneously hypertensive rats (SHR). Vascular thromboxane A2 generation was significantly enhanced by 49% in 5-week-old and by 117% in 15-week-old SHR as compared with age-matched Wistar-Kyoto rats (WKY). Thromboxane A2 generation was also significantly enhanced by 59% in the cultured vascular smooth muscle cells of SHR when compared with production in WKY. Vascular smooth muscle cells of SHR exhibited a significantly shortened doubling time (by 32%) and greater [3H]thymidine uptake (by 56%), as compared with those of WKY. OKY 046 (10(-5) M), a thromboxane synthase inhibitor, significantly tempered the rapid vascular smooth muscle cell growth in SHR by 9% for doubling time and by 10% for [3H]thymidine uptake. OKY 046 did not influence the doubling time of WKY. Conversely, a stable analogue of thromboxane A2 dose-dependently stimulated the [3H]thymidine uptake by vascular smooth muscle cells of WKY, and, at a concentration of 10(-5) M, shortened the doubling time of vascular smooth muscle cells of WKY by 11%, whereas it showed slight effects on SHR. These data indicate that vascular thromboxane A2 is involved in the regulatory mechanism of vascular smooth muscle cell growth and that enhanced vascular thromboxane A2 generation is partly responsible for the rapid proliferation of vascular smooth muscle cells of SHR. The alterations of vascular thromboxane production may be a key trait for genetic hypertension.

Thromboxane released from activated platelets and prostacyclin of the vessel wall may act as potent antagonistic modulators of platelet aggregability and coronary vascular tone. Therefore, urinary excretion of their major metabolites, 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F1 alpha, was studied in 16 patients presenting with prolonged angina at rest. The 10 patients whose condition did not improve under vigorous antianginal treatment within 48 hours exhibited higher thromboxane metabolite excretion than did the 6 patients who responded to therapy (2,208 +/- 1,542 versus 609 +/- 312 ng/g creatinine; p less than 0.001). Elevated values were also found in four of eight patients with sustained postinfarction angina. Enhanced thromboxane metabolite excretion was frequently associated with angiographic evidence of thrombus formation. When nine patients were restudied in a stable phase after 11 +/- 5 months, thromboxane metabolite excretion was consistently normal or high normal. Excretion of prostacyclin metabolites was not depressed in any patient but correlated weakly with thromboxane (r = 0.41). Thus, enhanced thromboxane production as an index of platelet activation may identify patients with active thrombus formation who could benefit most from platelet inhibitory treatment.

Platelet aggregation is known to be increased in diabetes mellitus, and the enhanced thromboxane production has been shown to be one of the causes of such abnormal platelet function. To investigate which step is activated in diabetic prostaglandin metabolism, three specific inhibitors of prostaglandin synthetases were used in this study, which were mepacrine, indomethacin and imidazole. Platelet aggregation induced by collagen was significantly increased accompanying enhanced thromboxane production in diabetics with proliferative retinopathy compared with age matched controls. Platelet aggregation in diabetics with proliferative retinopathy was less inhibited by the addition of each inhibitor compared with controls. However, there was no difference in inhibitory pattern of platelet aggregation among the three inhibitors. In addition, thromboxane production during aggregation in diabetics with proliferative retinopathy was significantly greater than that in controls by the addition of each inhibitor. Inhibitory patterns of thromboxane production did not differ among the addition of three inhibitors. It is concluded that enhanced thromboxane production resulting in enhanced platelet aggregation would be related to diabetic vascular complications. This abnormal prostaglandin production can be due to the activation of general steps in prostaglandin metabolism in diabetic platelets, not of a specific enzyme.

The appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells was investigated during 1 alpha,25-dihydroxyvitamin D-3-induced monocytic differentiation. 1 alpha,25-Dihydroxyvitamin D-3-treated HL-60 cells acquired the ability to convert [1-14C]arachidonic acid to thromboxane B2 and prostaglandin E2 during monocytic differentiation. The major cyclooxygenase product synthesized by the HL-60 cells after 3-4 days exposure to 1 alpha,25- dihydroxyvitamin D-3 (48 nM) was thromboxane B2 and its production was about 19-25-times higher than that of untreated HL-60 cells. The percent conversion of thromboxane B2 from [1-14C]arachidonic acid in the 1 alpha,25-dihydroxyvitamin D-3 (48 nM, 3 day exposure)-treated HL-60 cells was about 4.4% as compared to that (about 0.3%) of the untreated cells, whereas the percent conversion of thromboxane B2 from [1-14C]prostaglandin H2 in the 1 alpha,25-dihydroxyvitamin D-3-treated cell homogenate was about 22.4% as compared to that (about 13.6%) of the untreated cell homogenate. The stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on thromboxane B2 production from [1-14C]arachidonic acid and from [1-14C]prostaglandin H2 in HL-60 cells was inhibited by the addition of cycloheximide (1 microgram/ml). However, 1 alpha,25-dihydroxyvitamin D-3 (48 nM) did not significantly stimulate the arachidonic acid release either in HL-60 cells or in 1 alpha,25-dihydroxyvitamin D-3-induced cells. These results suggest that the stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on the thromboxane production in HL-60 cells was not due to the activation of phospholipase A2 but due to the induction of fatty acid cyclooxygenase and thromboxane synthetase activities. Thromboxane A2 actively produced during the monocytic differentiation of HL-60 cells could influence the cell adhesiveness of the monocyte-macrophage-differentiated cells.

Citalopram prevents serotonin (5-HT) uptake into platelets by blocking the serotonin reuptake transporter (SERT). Although some clinical data suggest that selective serotonin reuptake inhibitors (SSRIs) may affect haemostasis and thrombosis, these poorly-characterised effects are not well understood mechanistically and useful in vitro data is limited. We sought to determine whether the inhibitory effects of citalopram on platelets are mediated via its pharmacological inhibition of 5-HT transport. We quantified the inhibitory potency of (RS)-, (R)- and (S)-citalopram on platelet function. If SERT blockade is the primary mechanism for citalopram-mediated platelet inhibition, these potencies should show quantitative congruence with inhibition of 5-HT uptake. Our data show that citalopram inhibits platelet aggregation, adhesion and thromboxane production with no difference in potency between (R)- and (S)-isomers. By contrast, citalopram had a eudysmic ratio of approximately 17 (S > R) for SERT blockade. Furthermore, nanomolar concentrations of citalopram inhibited 5-HT uptake into platelets but had no effect on other platelet functions, which were inhibited by micromolar concentrations. Our data indicate that citalopram-induced inhibition of platelets in vitro is not mediated by blockade of 5-HT transport. This raises a new question for future investigation: by what mechanism(s) does citalopram inhibit platelets?

SummaryIn 13 type II hyperlipidemics (10 males and 3 females; mean age 50.2 ± 10.6 years), in 10 type IV hyperlipidemics (7 males and 3 females; mean age 51 ± 13.3 years) and in 23 healthy age-and sex-matched controls, the following parameters were measured: plasma cholesterol; plasma TG; plasma C-HDL; VLDL, separated in a preparative ultracentrifuge; C-LDL; Apo B, with immunoelectrophoretic method; platelet sensitivity to prostacyclin; TXB2 formation in PRP; TXB2 in serum.This study provides evidence for: 1. Reduced platelet sensitivity to prostacyclin, more evident in type II hyperlipidemia that provides an additional mechanism involved in increased platelet aggregation found in type II hyperlipidemia. 2. Enhanced TXB2 formation in PRP after thrombin stimulation (664.65 ± 142.18 pmol/108 platelets) only in type II hyperlipidemics and such enhanced formation was positively correlated to C-LDL (r = 0.53; p

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine- diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

L'agregation des plaquettes humaines induite par le facteur activant les plaquettes depend en partie de la production de thromboxane A2

We have studied the effect of amniotic fluid on thromboxane A 2 (TXA 2 ) production as an initial step in an evaluation of the role of this metabolite as the mediator of the pulmonary hypertension that accompanles perinatal aspiration. Term amniotic fluid enhanced platelet thromboxane B 2 (TXB 2 ) production in the presence of the aggregating agents thrombin and arachidonic acid, activity being confined to the lipid fraction. Compared with a baseline production of 1.4±0.45 pmol TXB 2 /10 6 platelets in response to thrombin (1 U/ml), unfractionated amniotic fluid or its lipid fraction enhanced TXB 2 production to 2.87±0.53 and 2.81±0.62 pmol, respectively (P 2 production was observed in amniotic fluid obtained at 15 to 17 weeks. Similar activity was observed with either adult or neonatal platelets. This thromboxane enhancing property of amniotic fluid appears to be distinct from its thrombin generating property. Following perinatal aspiration, in situ production of thrombin and proaggregatory TXA 2 could recruit more platelets, enhance local TXA 2 production, and be responsible for the platelet thrombi that have been documented at autopsy in the pulmonary microcirculation in infants with perinatal aspiration syndrome. (

• The influence of venous patching on luminal prostacyclin (PGI 2 ) and thromboxane A 2 (TBX) production of endarterectomized arteries was studied in a canine model. Fifteen dogs underwent bilateral common carotid endarterectomies. In each dog, one artery was closed primarily and the contralateral artery was closed by vein-patch angioplasty. At six and 12 weeks after operation, luminal prostanoid production from the venous patch, adjacent endarterectomized artery, and control artery and vein was measured by radioimmunoassay for 6-keto-prostaglandin F 1α and thromboxane B 2 . Venous patches underwent "incomplete" biochemical adaptation with decreased luminal production of PGI 2 compared with control and endarterectomized arteries. Thromboxane production from venous patches was increased compared with normal vein but similar to control and endarterectomized arteries. At six weeks, PGI 2 production of vein-patched arterial segments was increased compared with arteries closed primarily. These data indicate that vein-patch angioplasty favorably influences surface thrombogenicity of healing, endarterectomized arteries and support its use in reparative carotid surgery. ( Arch Surg 1989;124:490-493)