Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.     

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.     

Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade.Platelets are small discoid anuclear cell fragments that are key players in haemostasis – a frontline physiological response to acute tissue injury. Under basal conditions, platelets circulate at 150–400 × 10⁹ per litre and sequester a diverse array of bioactive molecules within their intracellular granules.¹ Upon tissue injury, however, vascular disruption triggers localised platelet deposition, activation and release of their granular contents. These platelet-released molecules (PRMs) further recruit and activate platelets, resulting in a multi-cellular aggregate that restricts blood loss. In conjunction, the coagulation cascade becomes activated, leading to thrombin generation which consolidates the growing thrombus by promoting further platelet activation and by catalysing fibrin formation.In addition to this critical role in haemostasis, platelets also participate in numerous non-haemostatic processes, including inflammation,² tissue repair,³ angiogenesis and lymphatic development.⁴ Platelets also promote tumour cell proliferation and metastasis via peri-cellular adhesion and signalling.^{5, 6, 7} Such pleiotropy is attributed to the array of molecules that are released from activated platelets.The reparative influence of platelets also has important clinical utility. For decades, ‘platelet concentrates'' have been applied to sites of injury to expedite the recovery of organs such as bone,⁸ skin⁹ and tendon.¹⁰ The beneficial effects of platelet concentrates are thought to be due to the many growth factors that are released from activated platelets.³ This reasoning, however, may be considered speculative given the lack of direct experimental evidence. Furthermore, little-to-no information exists about the platelet-derived peri-cellular signals that facilitate tissue repair.Platelets are normally restricted to the intravascular compartment. However, extravascular platelet accumulation can also occur under certain pathological conditions. One notable example of this is neurotrauma. Here, we find that exceptionally high levels of platelet activation occur in the brain after neurotrauma, thereby providing in-principle support that platelet products may influence neuronal survival. We therefore examined the influence of PRMs on injured primary cortical neurons. As neurons are post-mitotic, this approach allowed the elucidation of effects that were unrelated to proliferation. We find that PRMs selectively and potently reduce neuronal apoptosis via paracrine activation of the epidermal growth factor receptor (EGFR) and downstream activation of DNA-dependent protein kinase (DNA-PK) – a ubiquitous DNA repair enzyme. Strikingly, the same platelet-dependent mechanism also protects several non-neuronal human cancer cell types from chemotherapy-induced apoptosis. Thus, activated platelets trigger a potent and broad-acting paracrine signal that attenuates apoptosis.Our findings highlight a beneficial action of platelets that likely operates within the injured brain where existing knowledge about platelets has been limited to its haemostatic role. Interestingly, we also find that this underappreciated cytoprotective action of platelets may have implications in cancer where the association between higher platelet count and poorer patient prognosis has been well established.^{11, 12, 13, 14} In addition, this newly identified anti-apoptotic mechanism should encourage rationally designed improvements to the clinical use of platelet concentrates.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

A particulate arachidonate lipoxygenase in human blood platelets

The microsomal fraction of human platelets contains a lipoxygenase activity in addition to the thromboxane-synthesizing activity. The enzymatic activity was stimulated by tryptophan, but inhibited by catecholamine, methemoglobin, and hydroquinones.     

Activated platelets express IL-1 activity

Suspensions of washed human platelets express IL-1 activity after activation with agents such as thrombin, collagen, ADP, or epinephrine as judged by the ability of the platelet suspensions to support the growth of a T cell line, D10.G4.1, which exhibits a growth requirement for IL-1. Unactivated platelets express little IL-1 activity. The IL-1 activity expressed by activated platelets appears to be entirely associated with the platelet surface. No IL-1 activity was detected in supernatants derived from suspensions of activated platelets. A mAb specific for IL-1 beta inhibited 90% of the activity expressed by thrombin-activated platelets, whereas a mAb specific for IL-1 alpha inhibited approximately 20% of the activity. A control mAb was without an effect. These results indicate that activated platelets express surface-associated IL-1 activity. Platelet surface IL-1 may provide a mechanism for altering in an extremely localized and rapid manner the properties of IL-1 responsive cells with which platelets come in direct contact during processes of inflammation and vessel wall damage.     

Cigarette smoking stimulates lipoxygenase but not cyclooxygenase pathway in platelets

Male rats were exposed to freshly generated cigarette smoke once daily for 4 to 8 weeks. Inhalation of smoke was verified by elevated level of carboxyhemoglobin. Arachidonate metabolism through lipoxygenase and cyclooxygenase pathways in platelets was determined. Cigarette smoking increased 12-lipoxygenase activity significantly without affecting the cyclooxygenase pathway. In view of platelet-leukocyte interactions and potent chemotactic activity of 12-HETE for aortic smooth muscle cell migration, increased 12-lipoxygenase activity may predispose individuals to atherosclerosis, thromboembolism and emphysema commonly found in smokers.     

Regulation of arachidonate metabolism via lipoxygenase and cyclo-oxygenase by 12-HPETE, the product of human platelet lipoxygenase.

Circular dichroism and resonance Raman spectra of the cluster anion: [Cu(II)₆Cu(I)₈(D-Penicillamine)₁₂Cl]^5? enable the assignment of the S(mercaptide)→Cu(II) charge transfer transition to a band lying at 18250 cm^?1. The resonance Raman spectra compare with that of copper-diethyldithiocarbamate obtained previously. The implications of both series of data upon the resonance Raman spectra of blue copper proteins and the assignment of the CuS(cys) stretching mode are pointed out.     

1-phenyl-3-pyrazolidone: an inhibitor of cyclo-oxygenase and lipoxygenase pathways in lung and platelets.

1-phenyl-3-pyrazolidone (phenidone) is a commercially available reagent used in the photographic industry. When tested as an inhibitor of arachidonic acid metabolsim in platelets and lungs it was found to be effective against both the cyclo-oxygenase and lipoxygenase pathways. It is suggested that this compound may be a useful pharmacological tool.     

GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium.

GMP-140 is a membrane glycoprotein located in secretory granules of platelets and endothelium. When these cells are activated by agonists such as thrombin, GMP-140 is rapidly translocated to the plasma membrane. GMP-140, along with ELAM-1 and the peripheral lymph node homing receptor, defines the selectin family of structurally related molecules that regulate interactions of leukocytes with the blood vessel wall. Each of these molecules contains an N-terminal lectin-like domain, followed by an EGF-like region, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The genomic structures of the selectins suggest that they arose by duplication and modification of exons encoding specific structural domains. GMP-140 is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to endothelium at regions of tissue injury as well as platelet-leukocyte interactions at sites of inflammation and hemorrhage. Like other leukocyte adhesion molecules, GMP-140 may also participate in pathologic inflammation, thrombosis, and tumor metastasis. Confirmation of such pathologic roles may lead to design of new drugs that block adhesive receptor function in human disease.     

Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.     

Platelet lipoxygenase-dependent oxygen burst. Evidence for differential activation of lipoxygenase in intact and disrupted human platelets

The metabolism of arachidonic acid in platelets by both cyclooxygenase and lipoxygenase involves the rapid consumption of molecular oxygen. However, selective inhibition of cyclooxygenase completely abolishes the arachidonate-induced oxygen burst in intact platelets. This is in contrast to platelet lysates, in which approximately 50% of the arachidonate-induced oxygen burst remains detectable following inhibition of cyclooxygenase with acetylsalicylic acid. This lipoxygenase oxygen burst is blocked by preincubation of the platelets with ETYA, which inhibits both cyclooxygenase and lipoxygenase. In cell-free 100000 x g supernatants of platelet lysates, which contain only lipoxygenase activity, arachidonate induces an oxygen burst which is not blunted by preincubation with aspirin but is completely abolished by preincubation with ETYA. The finding of a lipoxygenase-dependent oxygen burst in platelet lysates but not in intact platelet suspensions suggests differential activation or differential availability of platelet lipoxygenase in intact and disrupted platelets. This was confirmed by a 5 min lag in the generation of [14C]HETE (the major lipoxygenase product) from [14C]arachidonic acid in intact platelets, but an almost immediate initiation of [14C]HETE production in platelet lysates. In contrast, the synthesis of [14C]thromboxane B2 (the major cyclooxygenase product) from [14C]arachidonic acid began immediately in both intact and disrupted platelet preparations and peaked within 5 min. These observations provide new insight into factors controlling platelet hydroxy acid production and help to explain the nature of the platelet oxygen burst.     

Altered lipoxygenase metabolism and decreased glutathione peroxidase activity in platelets from selenium-deficient rats

Washed platelets from selenium-deficient and control rats were incubated with [1-¹⁴C]-arachidonic acid and the lipoxygenase and cyclooxygenase products were identified by gas chromatography/mass spectrometry. Platelets from selenium-deficient rats showed a three to four-fold increased synthesis of the lipoxygenase-derived isomeric trihydroxy fatty acids, 8,9,12-trihydroxy-5,10,14-eicosatrienoic acid and 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid. A major reduction in glutathione peroxidase activity was also observed in platelets from deficient rats. These results support the interpretation that these trihydroxy fatty acids arise from breakdown of the primary platelet lipoxygenase product $\text{L}$-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) under conditions in which its reduction to the $\text{L}$-12-hydroxy product (12-HETE) by a selenium-dependent glutathione peroxidase is limited. Further-more, these results indicate a specific function for selenium in platelet metabolism of essential fatty acids.     

Activated platelets contribute importantly to myocardial reperfusion injury

Platelets become activated during myocardial infarction (MI), but the direct contribution of activated platelets to myocardial reperfusion injury in vivo has yet to be reported. We tested the hypothesis that activated platelets contribute importantly to reperfusion injury during MI in mice. After 30 min of ischemia and 60 min of reperfusion, P-selectin knockout mice had a significantly smaller infarct size than that of wild-type mice (P < 0.05). Platelets were detected by P-selectin antibody in the previously ischemic region of wild-type mice as early as 2 min postreperfusion after 45 min, but not 20 min, of ischemia. The appearance of neutrophils in the heart was delayed when compared with platelets. Flow cytometry showed that the number of activated platelets more than doubled after 45 min of ischemia when compared with 20 min of ischemia or sham treatment (P < 0.05). Platelet-rich or platelet-poor plasma was then transfused from either sham-operated or infarcted mice after 45 and 10 min of ischemia-reperfusion to mice undergoing 20 and 60 min of ischemia-reperfusion. Infarct size was increased by threefold and platelet accumulation was remarkably enhanced in mice treated with wild-type, MI-activated platelet-rich plasma but not in mice receiving either platelet-poor plasma from wild types or MI-activated platelet-rich plasma from P-selectin knockout mice. In conclusion, circulating platelets become activated early during reperfusion and their activation depends on the duration of the preceding coronary occlusion and is proportional to the extent of myocardial injury. Activated platelets play an important role in the process of myocardial ischemia-reperfusion injury, and platelet-derived P-selectin is a critical mediator.     

Identification of four major classes of sulfhydryl groups in human blood platelets. Ferricyanide titration of spin-labeled platelets.

Human blood platelets have been labeled with the sulfhydryl-specific spin labels, 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl and 3-maleimido-2,2,5,5-tetramethylpyrrolidine-1-oxyl. First, the ESR spectra of platelets labeled with either reagent revealed two classes of sulfhydryl groups, a mobile class and an immobile class. Second, when spin-labeled platelets were titrated with high concentrations of potassium ferricyanide (greater than 10(-3) M), there was a decrease in the peak heights of the mobile class of sulfhydryl groups due to dipole-dipole exchange. Third, plots of peak heights of the mobile class versus ferricyanide concentration revealed three classes of mobile sulfhydryl groups compared to a single immobile class. This technique may be used to show the relative locations of spin-labeled groups on cell surfaces.     

Preferential binding of platelets to monocytes over neutrophils under flow

This study was undertaken to systematically investigate the binding kinetics of platelet recruitment by monocytes relative to neutrophils in bulk suspensions subjected to shear as well as the molecular requirements of leukocyte-platelet binding. Hydrodynamic shear-induced collisions augment the proportion of monocytes with adherent platelets more drastically than that of neutrophils with bound platelets. These heterotypic interactions are further potentiated by platelet activation with thrombin or to a lesser extent by monocyte stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Monocyte-platelet heteroaggregation increases with increasing shear rate and shear exposure time. Platelet P-selectin binding to monocyte P-selectin-glycoprotein-ligand-1 is solely responsible for maximal platelet adhesion to unstimulated monocytes in shear flow. However, the enhanced platelet binding to fMLP-treated monocytes involves a sequential two-step process, wherein P-selectin-PSGL-1 interactions are stabilized by CD18-integrin involvement. Blocking platelet alpha(IIb)beta(3) or monocyte beta(1)-integrin function had no effect. This study underscores the preferential recruitment of platelets by monocytes relative to neutrophils in shear flow, and demonstrates that the shear environment of the vasculature coupled to the state of cell activation modulates the dynamics and molecular constituents mediating monocyte-platelet adhesion.     

Activated human monocytes oxidize low-density lipoprotein by a lipoxygenase-dependent pathway

Monocyte-mediated oxidation of low-density lipoprotein (LDL) converts the lipoprotein to a potent cytotoxin. The oxidation process requires monocyte activation and requires superoxide anion since it can be blocked by superoxide dismutase. In this study, the requirement for lipoxygenase activity is shown, in that 1) inhibitors of lipoxygenase prevent the alteration of LDL, 2) copper (II) (3,5-diisopropylsalicylic acid), an agent shown to enhance lipoxygenase activity in a cell-free system, similarly enhances monocyte-mediated LDL alteration, and 3) the (3,5-diisopropylsalicylic acid)-enhanced monocyte-mediated modification of LDL can be completely blocked by inhibitors of lipoxygenase or by superoxide dismutase. These data suggest an integral role for monocyte lipoxygenase in the generation by activated monocytes of the extracellular superoxide anion that participates in the oxidation of LDL and the conversion of LDL to a cytotoxin. Monocyte-modified LDL may be a mediator in tissue damage that accompanies atherosclerosis or occurs at sites of inflammation.     

Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB2 and HHT formation at concentrations of 8 microM and below, and at 50 microM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.