Ji-Yoon Noh

Lysophosphatidic Acid Induces Thrombogenic Activity Through Phosphatidylserine Exposure and Procoagulant Microvesicle Generation in Human Erythrocytes

Objective—Although erythrocytes have been suggested to play a role in blood clotting, mediated through phosphatidylserine (PS) exposure and/or PS-bearing microvesicle generation, an endogenous substance that triggers the membrane alterations leading to a procoagulant activity in erythrocytes has not been reported. We now demonstrated that lysophosphatidic acid (LPA), an important lipid mediator in various pathophysiological processes, induces PS exposure and procoagulant microvesicle generation in erythrocytes, which represent a biological significance resulting in induction of thrombogenic activity. Methods and Results—In human erythrocytes, LPA treatment resulted in PS exposure on remnant cells and PS-bearing microvesicle generation in a concentration-dependent manner. Consistent with the microvesicle generation, scanning electron microscopic study revealed that LPA treatment induced surface changes, alteration of normal discocytic shape into echinocytes followed by spherocytes. Surprisingly, chelation of intracellular calcium did not affect LPA-induced PS exposure and microvesicle generation. On the other hand, protein kinase C (PKC) inhibitors significantly reduced PS exposure and microvesicle generation induced by LPA, reflecting the role of calcium-independent PKC. Activation of PKC was confirmed by Western blot analysis showing translocation of calcium-independent isoform, PKC&zgr;, to erythrocyte membrane. The activity of flippase, which is important in the maintenance of membrane asymmetry, was also inhibited by LPA. Furthermore, LPA-exposed erythrocytes actually potentiated the thrombin generation as determined by prothrombinase assay and accelerated the coagulation process initiated by recombinant human tissue factor in plasma. The adherence of erythrocytes to endothelial cells, another important feature of thrombogenic process, was also stimulated by LPA treatment. Conclusion—These results suggested that LPA-exposed erythrocytes could make an important contribution to thrombosis mediated through PS exposure and procoagulant microvesicle generation.

Silver nanoparticles enhance thrombus formation through increased platelet aggregation and procoagulant activity

Abstract Despite the wide use of silver nanoparticles (nano Ag), its toxicity still remains poorly understood. In this report, nano Ag induced an increase in platelet aggregation and procoagulant activation which are the key contributors to thrombotic diseases. In freshly isolated human platelets, nano Ag induced platelet aggregation and procoagulant activation evident by increased phosphatidylserine exposure and thrombin generation. Interestingly, the sub-threshold level of thrombin enhanced nano Ag-induced platelet activation significantly indicating that the prothrombotic effects of nano Ag might be further potentiated in activated platelets. An increase in intracellular calcium mediated nano Ag induced platelet activation and P-selectin expression, and serotonin release was also enhanced by nano Ag. Consistent with the in vitro results, exposure to nano Ag (0.05–0.1 mg/kg i.v. or 5–10 mg/kg intratracheal instillation) in vivo enhanced venous thrombus formation, platelet aggregation, and phosphatidylserine externalization ex vivo in rats suggesting that nano Ag, indeed, does enhance thrombus formation through platelet activation.

Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.

Summary.  Background: Doxorubicin (DOX) is a widely used anticancer drug for solid tumors and hematologic malignancy, but its active use is hampered by serious adverse effects, including thrombocytopenia. Although bone marrow toxicity of DOX has been suggested to be the sole mechanism underlying the reduced platelet counts, the direct effects of DOX on platelets have never been examined. Objective: Here, we investigated the DOX‐induced platelet cytotoxicity and its underlying mechanism in an effort to elucidate the contribution of platelet cytotoxicity to DOX‐induced thrombocytopenia. Results: In freshly isolated human platelets, DOX induced platelet cytotoxicity in a time‐dependent and concentration‐dependent manner. Reactive oxygen species (ROS) generation, decreased glutathione levels and subsequent protein thiol depletion were shown to underlie the DOX‐induced platelet cytotoxicity. Conspicuously, DOX‐treated platelets displayed apoptotic features such as caspase‐3 activation, reduced mitochondrial transmembrane potential, and phosphatidylserine exposure. Decreased glutathiolation of procaspase‐3 was shown to be a link between protein thiol depletion and caspase‐3 activation. It is of note that DOX‐mediated platelet cytotoxicity was significantly enhanced by shear stress, a common complicating factor in cancer patients. These in vitro results were further confirmed by an in vivo animal model, where administration of DOX induced a platelet count decrease, ROS generation, caspase‐3 activation, protein thiol depletion, and damaged platelet integrity. Conclusion: We demonstrated that DOX can directly induce platelet cytotoxicity through ROS generation, decreased glutathione levels, and protein thiol depletion. We believe that this study provides important evidence for the role of DOX‐induced platelet cytotoxicity in the development of thrombocytopenia in DOX‐treated patients.

Low-Level Mercury Can Enhance Procoagulant Activity of Erythrocytes: A New Contributing Factor for Mercury-Related Thrombotic Disease

Background Associations between cardiovascular diseases and mercury have been frequently described, but underlying mechanisms are poorly understood. Objectives We investigate the procoagulant activation of erythrocytes, an important contributor to thrombosis, by low-level mercury to explore the roles of erythrocytes in mercury-related cardiovascular diseases. Methods We used freshly isolated human erythrocytes and ex vivo and in vivo thrombosis models in rats to investigate mercury-induced procoagulant activity. Results Prolonged exposure to low-dose mercuric ion (Hg2+; 0.25–5 μM for 1–48 hr) induced erythrocyte shape changes from discocytes to echinocytes to spherocytes, accompanied by microvesicle (MV) generation. These MVs and remnant erythrocytes expressed phosphatidylserine (PS), an important mediator of procoagulant activation. Hg2+ inhibited flippase, an enzyme that recovers PS into the inner leaflet of the cell membrane, and activated scramblase, an enzyme that alters lipid asymmetry in the cell membrane. Consistent with these activity changes, Hg2+ increased intracellular calcium and depleted ATP and protein thiol. A thiol supplement reversed Hg2+-induced MV generation and PS exposure and inhibited the increase in calcium ion (Ca2+) and depletion of ATP, indicating that free-thiol depletion was critical to Hg2+-mediated procoagulant activity. The procoagulant activity of Hg2+-treated erythrocytes was demonstrated by increased thrombin generation and endothelial cell adhesion. We further confirmed Hg2+-mediated procoagulant activation of erythrocytes in ex vivo and in vivo rat thrombosis models, where Hg2+ treatment (0.5–2.5 mg/kg) increased PS exposure and thrombus formation significantly. Conclusion This study demonstrated that mercury could provoke procoagulant activity in erythrocytes through protein-thiol depletion–mediated PS exposure and MV generation, ultimately leading to enhanced thrombosis.

Low level of lead can induce phosphatidylserine-exposure and erythrophagocytosis:A new mechanism underlying lead-associated anemia

Anemia is probably one of the most well-known toxic effects of lead. Previously, lead-induced anemia was considered to be from the inhibition of δ-aminolevulinic acid dehydratase participating in the heme biosynthesis. However, little is known whether lead could affect the destruction of erythrocyte, another important factor for anemia. In the present study, we demonstrated that lead could accelerate the splenic sequestration of erythrocytes through phosphatidylserine (PS) exposure and subsequently increased erythrophagocytosis. In freshly isolated human erythrocytes, Pb(2+)- induced PS exposure at relatively low concentrations (∼0.1 μM) by inhibiting flippase, a key aminophospholipid translocase for the maintenance of PS asymmetry and adenosine triphosphate depletion appeared to underlie this phenomenon. Abnormal shape changes of erythrocytes and microvesicle generation and other triggers for the erythrophagocytosis were also observed in the Pb(2+)-exposed erythrocytes. In vitro data showed that human macrophage indeed recognized and phagocytosis PS-exposed erythrocytes. In good accordance with these in vitro results, the oral administration of Pb(2+) increased PS exposure on erythrocytes in rat in vivo. In addition, reduction of hematocrit and hemoglobin and increased spleen weight were observed along with enhanced splenic sequestration of erythrocytes in the rats exposed to Pb(2+) subchronically for 4 weeks through drinking water. In conclusion, these results suggest that Pb(2+)-induced anemia may be explained at least in part by increased PS exposure on erythrocytes, erythrophagocytosis, and splenic sequestration.

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

Anti-Inflammatory and Analgesic Activities of SKLJI, a Highly Purified and Injectable Herbal Extract of Lonicera japonica

The parenteral route has many merits over the oral route, including greater predictability, reproducibility of absorption, and rapid drug action, but injectable phytomedicines are uncommon due to protein precipitating tannin and hemolytic saponin components. In this study, in an effort to develop a safe injectable analgesic phytomedicine, we prepared a tannin and saponin-free Lonicera japonica extract, SKLJI, through fractionation and column purification, and evaluated its anti-inflammatory and analgesic activities in in vivo experimental models of inflammation and pain. The removal of tannin and saponin resulted in loganin and sweroside-enriched SKLJI and it showed reduced hemolysis and protein precipitation. In efficacy tests, SKLJI inhibited croton oil- and arachidonic acid-induced ear edema, acetic acid-induced writhing, and carrageenan-induced rat hind paw hyperalgesia. Inhibition of cylcooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and 5-lipoxyfenase (5-LO) activities by SKLJI appeared to be the mechanism underlying anti-inflammatory and analgesic efficacy. Loganin and sweroside also showed anti-inflammatory and analgesic activities, suggesting that they might be active principles in the efficacy of SKLJI. These results suggest that SKLJI is a viable candidate for a new anti-inflammatory and analgesic phytomedicine that can be administered by the parenteral route.

Lead-induced procoagulant activation of erythrocytes through phosphatidylserine exposure may lead to thrombotic diseases

Lead (Pb) is a ubiquitous heavy metal pollutant in various environmental media, especially in food and drinking water. In human blood, about 95% of lead is associated with erythrocytes, suggesting that erythrocytes could be an important target of lead toxicity in the cardiovascular system. Recent studies suggested that erythrocytes could contribute to blood coagulation via phosphatidylserine (PS) exposure and resultant procoagulant activation. We investigated the effects of lead on the procoagulant activity of erythrocytes using in vitro human erythrocyte and in vivo rat models. In a flow cytometric analysis, lead (Pb2+) enhanced PS exposure on human erythrocytes in a concentration- and time-dependent manner. The concentration of lead (1-5 microM) used in the current investigation is well within the ranges observed in blood from lead-exposed populations. PS exposure by lead appeared to be mediated by increased intracellular calcium levels as shown by 19F-NMR and intracellular ATP depletion. Consistent with these findings, the activity of scramblase, which is important in the induction of PS exposure, was enhanced, whereas the activity of flippase, which translocates exposed PS to inner membrane, was inhibited by lead treatment. Furthermore, lead-exposed erythrocytes increased thrombin generation as determined by a prothrombinase assay and accelerated the coagulation process initiated by tissue factor in plasma. These procoagulant activations by lead were also confirmed in vivo. Administration of lead significantly enhanced PS exposure on erythrocytes and, more importantly, elevated thrombus formation in a rat venous thrombosis model. These results suggest that lead exposure can provoke procoagulant activity in erythrocytes by PS exposure, contributing to enhanced clot formation. These data will provide new insights into the mechanism of lead-induced cardiovascular diseases.

U-shaped Dose Response in Vasomotor Tone: A Mixed Result of Heterogenic Response of Multiple Cells to Xenobiotics

U-shaped response has been frequently encountered in various biological areas including epidemiology, toxicology, and oncology. Despite its frequent observation, the theory of U-shaped response has been crippled by the lack of a robust mechanism underlying and incomplete in vitro and in vivo correlation. In the present study, a novel mechanism is provided for a U-shaped response, based on the findings of agonist-induced vasomotor tone change affected by menadione (MEN) (synthetic vitamin K(3)), a reactive oxygen species generator, and arsenic, an environmental pollutant, which showed typical U-shaped responses in both in vitro aortic contractile response and in vivo blood pressure. U-shaped responses by MEN and arsenic were a combined result from heterogenic susceptibilities and responses of multiple target cells composing blood vessels, that is, endothelium and smooth muscle. Notably, endothelium, a regulator of vasomotor tone, was primarily affected by low-dose stimuli, whereas smooth muscle, an effector of vascular contraction, was affected later by high-dose. The dysfunction of smooth muscle was produced by high-dose MEN-induced hydrogen peroxide, resulting in the attenuation of vascular contractile reactivity, whereas low-dose MEN-induced superoxide led to the quenching of vasodilatory nitric oxide in endothelial cells, resulting in the enhancement of vasoconstriction. This mechanistic theory, the difference in susceptibilities and responses to a common stimulus between regulator and effector components of a system, could give a new insight into the explanation of various U-shaped responses and provide a new evidence for the need of the risk assessment of toxicants with a wider dose range.

Doxorubicin-induced platelet procoagulant activities: an important clue for chemotherapy-associated thrombosis.

Thrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported; yet, the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle (MP) generation. Consistently, DOX-treated platelets and generated MPs induced thrombin generation, a representative marker for procoagulant activity. DOX-induced PS exposure appeared to be from intracellular Ca²⁺ increase and ATP depletion, which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (Δψ), cytochrome c release, Bax translocation, and caspase-3 activation. A Ca²⁺ chelator ethylene glycol tetraacetic acid, caspase inhibitor Q-VD-OPh, and antioxidants (vitamin C and trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that Ca²⁺, apoptosis, and reactive oxygen species (ROS) were involved in DOX-enhanced procoagulant activity. Importantly, rat in vivo thrombosis model demonstrated that DOX could manifest prothrombotic effects through the mediation of platelet procoagulant activity, which was accompanied by increased PS exposure and Δψ dissipation in platelets.