Platelet Lipoxygenase Pathway Research Articles

Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion

Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

12-Hydroxyeicosatetraenoic acid upregulates P-selectin-induced tissue factor activity on monocytes

12-Hydroxyeicosatetraenoic acid (12-HETE), a product of the platelet lipoxygenase pathway, amplifies tissue factor expression by P-selectin-stimulated monocytes in a time- and dose-dependent fashion. The same effect is observed when monocytes are incubated with Chinese hamster ovary cells transfected with the P-selectin cDNA. Both 5-HETE and leukotriene C 4 are inactive in this system. Furthermore, the effect is not dependent on non-specific monocyte adhesion, since monocytes incubated with CHO cells expressing E-selectin do not express tissue factor, either in the presence or in the absence of 12-HETE. These results show that 12-HETE is a cofactor for the expression of tissue factor by monocytes.

Increased lipopolysaccharide-induced tissue factor activity and tumour necrosis factor production in monocytes after intake of aspirin: possible role of prostaglandin E2.

To determine how aspirin intake might influence lipopolysaccharide (LPS)-induced tissue factor (TF) activity and tumour necrosis factor (TNF) in human blood monocytes, we collected blood before and at various times after intake of 300 mg aspirin in 25 healthy volunteers. Aspirin intake reduced LPS-induced thromboxane B2 and PGE2 production in whole blood by 50% and 65% respectively, measured 1 h after aspirin intake. Subsequently, a 95% rise in LPS-induced TF activity in monocytes was seen as compared to a 26% rise in TNF. The rise in TF activity was maximal within 1 h after aspirin intake and no further rise was observed 3, 4 or 24 h after aspirin intake. In contrast, TF activity induced by incubating whole blood in the absence of LPS fell rapidly after the intake of aspirin. In separate experiments, a dose-dependent inhibition by PGE2 was observed in LPS-induced TF activity in monocytes. It is proposed that the increased LPS-induced TF activity and TNF production following aspirin intake may be due to suppressed PGE2 formation. The more pronounced rise in TF activity compared to TNF production may be due to an enhancement of the platelet lipoxygenase pathway that has been shown to be important for LPS-induced TF activity in monocytes.

Transcellular lipoxygenase metabolism between monocytes and platelets.

We have examined the effects of co-culture and in vitro co-stimulation on lipoxygenase metabolism in monocytes and platelets. Monocytes were obtained from the peripheral blood of normal volunteers by discontinuous gradient centrifugation and adherence to tissue culture plastic. Platelets were obtained from the platelet-rich plasma of the same donor. When 10(9) platelets and 2.5 x 10(6) monocytes were co-stimulated with 1 microM A23187, these preparations released greater quantities of 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid, 5(S),12-(S)dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, and leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic (LTC4) when compared with monocytes alone. Release of arachidonic acid, 5-HETE, delta 6-trans-LTB4, and delta 6-trans-12-epi-LTB4 from monocytes was decreased in the presence of platelets. A dose-response curve was constructed and revealed that the above changes became evident when the platelet number exceeded 10(7). Dual radiolabeling experiments with 3H- and 14C-arachidonic acid revealed that monocytes provided arachidonic acid, 5-HETE, and LTA4 for further metabolism by the platelet. Monocytes did not metabolize platelet intermediates detectably. In addition, as much as 1.2 microM 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid and 12(S)-hydroperoxy-10-trans-5,8,14-cis-eicosatetraenoic acid had no effect on monocyte lipoxygenase metabolism. Platelets were capable of converting LTA4 to LTC4, but conversion of LTA4 to LTB4 was not detected. We conclude that the monocyte and platelet lipoxygenase pathways undergo a transcellular lipoxygenase interaction that differs from the interaction of the neutrophil and platelet lipoxygenase pathways. In this interaction monocytes provide intermediate substrates for further metabolic conversion by platelets in an unidirectional manner.

Altered platelet lipoxygenase activity in patients with low selenium.

1. Arachidonic acid metabolism through the lipoxygenase pathway in platelets was investigated in 11 patients with low selenium (Se) nutritional status and compared with 14 patients with a normal Se status. 2. Plasma and whole blood Se levels, as well as plasma, whole blood and platelet glutathione peroxidase (GSHPx) activity, were measured in both groups and found to be significantly lower in the group with low Se status. 3. Studies on [14C]arachidonic acid stimulated platelets demonstrated that reduced conversion of the active metabolite 12-hydroperoxyeicosatetraenoic acid to 12-hydroxyeicosatetraenoic acid occurred in patients with low Se nutrition. 4. Changes in [14C]arachidonic acid metabolism are attributed to reduced activity of the seleno-enzyme GSHPx. The effects of other factors cannot be entirely excluded.

Die Wirkung der Influenza-Virus-Infektion auf die Arachidons�ure-Kaskade von M�usethrombozyten

Prostaglandins (PGs) are essential for many physiological and pathological processes. As they are not stored in tissue, their presence and actions therefore result from de novo synthesis and release. Although platelets themselves appear to have the ability to synthesize TxA2, PGD2, arachidonic acid may also be metabolized in the lipoxygenase pathway in platelets, producing 12-hydroperoxy/12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HPETE/12-HETE). CFLP mice were infected intranasally with A/H3N2/Hong Kong (1/68) influenza virus. Platelets were isolated from the control (saline treated) and infected mice 3-13 days after virus application. Platelets were isolated from the diluted arterial blood of the mice. Metabolites of arachidonate cascade were determined using 1-14C-arachidonic acid (2035 MBq/mM spec. act.) as substrate. All incubations were carried out in TC Medium 199 (pH 7.4) at 37 degrees C for 10 min. Radiolabelled products were separated and quantitatively determined. The synthesis of TxA2 in the platelets of animals was found to be significantly increased 7 days after the virus infection. The 12-hydroxy-heptadecatrienoic acid level was higher on the 10th and 13th days of infection, as were the products of the lipoxygenase pathway.

Deficient induction of leukotriene synthesis in human neutrophils by lipoxygenase-deficient platelets

The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N- formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase- deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5- lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5-lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12- hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities.

Deficient Induction of Leukotriene Synthesis in Human Neutrophils by Lipoxygenase-Deficient Platelets

The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase-deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5-lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5-lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12-hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities.

Platelet release reaction and aggregation induced by canatoxin, a convulsant protein: evidence for the involvement of the platelet lipoxygenase pathway

Canatoxin is a toxic protein isolated from Canavalia ensiformis seeds. It induces death preceded by convulsions of spinal cord origin and also produces in vitro aggregation of platelets in rabbit, human and guinea-pig plasma. The aggregating effect is dose-dependent at nanomolar concentrations. Rabbit platelets pretreated with canatoxin became refractory to a second exposure to this protein or to collagen, but were still responsive to ADP, Paf-acether or arachidonic acid. [14C]-5-hydroxytryptamine was released from pre-labelled platelets on stimulation with canatoxin. Washed rabbit platelets, but not thrombin-degranulated ones, aggregated on stimulation with canatoxin provided that fibrinogen was added before the toxin. Canatoxin's pro-aggregating activity was inhibited by mepacrine, EDTA, caffeine, prostacyclin, adenosine monophosphate and also by the ADP scavenger system, creatine phosphokinase/creatine phosphate. Furthermore, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C), eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA) were potent inhibitors of canatoxin-induced aggregation. In contrast, no inhibition was seen with indomethacin. The data indicate that canatoxin is mainly a release-reaction-promoting agent, being devoid of any direct aggregating activity. Thus the aggregation is totally dependent on the release of ADP. Furthermore, canatoxin-induced platelet activation is probably dependent on platelet phospholipase A2 and lipoxygenase activity but is not dependent on cyclo-oxygenase products or the release of Paf-acether.

Esterification of monohydroxyfatty acids into the lipids of a macrophage cell line

Cells of a mouse macrophage-like tumor cell line, J774.2, were incubated with 0.6μM radiolabeled mono- and di-hydroxyfatty acids. Monohydroxyfatty acid products of the neutrophil and platelet lipoxygenase pathways (5-HETE, 15-HETE, and 12-HETE) were rapidly taken up (42–64% of the counts cell associated at 1 min) and esterified into triglycerides and phospholipids. 5-HETE and 12-HETE were found in triglycerides and distributed among phospholipid classes while 50% of added 15-HETE was esterified into phosphatidyl inositol. Treatment of phospholipids from cells incubated with 5-HETE, 12-HETE, and 15-HETE with phospholipase A 2 resulted in release of the respective monohydroxyfatty acid. HHT, a monohydroxyfatty acid product of the cyclooxygenase pathway, was taken up and esterified more slowly than the lipoxygenase products. In addition, HHT was not released when the phospholipids from cells incubated with HHT were treated with phospholipase A 2. LTB 4, a dihydroxyfatty acid product of neutrophil lipoxyegnase, was not taken up by J774.2 cells. The unique patterns of uptake and intracellular distribution of the different monohydroxyfatty acids suggests that the enzymes involved in the esterification of these compounds have substrate specificity and may also relate to the specific biologic effects of the compounds.

Role of glutathione peroxidase and hexose monophosphate shunt in the platelet lipoxygenase pathway.

Role of glutathione peroxidase and hexose monophosphate shunt in the platelet lipoxygenase pathway.

Deficiency of platelet lipoxygenase activity in myeloproliferative disorders.

The myeloproliferative disorders are characterized by frequent bleeding and thrombotic complications, which have been attributed to abnormal platelet function. In 24 of 60 patients studied, reduced activity of the platelet lipoxygenase pathway for oxygenation of arachidonic acid was revealed by a new direct assay. This assay measured arachidonic acid-induced oxygen consumption in platelets preincubated with aspirin to block cyclooxygenase activity. Patients with secondary polycythemia or thrombocytosis had normal lipoxygenase activity . In the cells of seven of eight patients with lipoxygenase deficiency arachidonic acid induced increased synthesis of thromboxane, the major cyclooxygenase product. Nevertheless, patients with deficient lipoxygenase activity tended to have episodes of hemorrhage rather than thrombosis. Bleeding complications occurred in 67 per cent of patients with lipoxygenase deficiency, but in only 19 per cent of those with normal lipoxygenase activity (P less than 0.001). In contrast, 13 per cent of lipoxygenase-deficient patients, but 31 per cent of patients with other myeloproliferative disorders, had thromboembolic complications. Measurement of platelet lipoxygenase activity may be of diagnostic value in distinguishing myeloproliferative disorders from secondary thrombocytosis or polycythemia. Lipoxygenase deficiency may prove to be a useful natural model for investigating the role of lipoxygenase products in hemostasis.

A Simple Method for Estimation of Lipoxygenase and Cyclo-Oxygenase Pathways in Human Platelets - the Use of Thiobarbituric Acid Reaction

To develop a simple method for estimation of platelet lipoxygenase (PLO) and cyclo-oxygenase (PCO) pathways, the arachidonic acid (AA) metabolism of human platelet was investigated under various experimental conditions by the use of the thiobarbituric acid (TBA) reaction and a radioisotope technique. A TBA-reactive substance different from malondialdehyde (MDA) via PCO pathway was detected and shown to be derived from the PLO pathway. Since the optimal pH and time course of its formation were different from those of MDA formation via PCO pathway, PLO and PCO pathways were estimated by quantitating the TBA-reactive substances produced by the incubation of AA either with aspirin-treated platelets or with untreated ones, respectively, each under optimal conditions. Normal values expressed in terms of nmol MDA/10(8) platelets were 1.17 +/- 0.34 (M +/- SD, n = 31) and 0.79 +/- 0.15 (n = 31) for PLO and PCO pathways respectively.

Isolation of a new lipoxygenase metabolite of arachidonic acid, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid from human platelets

Washed human platelets incubated with 1- 14C -arachidonic acid (1mM) produced a new metabolite which migrated on thin layer chromatography close to thromboxane B 2, but which was identified by mass spectrometry as a trihydroxy fatty acid. The mass spectrum was consistent with the assigned structure, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid (THETE). Platelet THETE synthesis from arachidonate was not inhibited by preincubation with aspirin or indomethacin but was blocked by 5,8,11,14-eicosatetraynoic acid. Therefore, THETE appears to arise via the platelet lipoxygenase pathway rather than via the prostaglandin cyclooxygenase. Two proposed structures, including a novel dihydro-hydroxy-pyran cyclic intermediate, which could give rise to THETE are presented.