Effect of Lead(IV) Acetate on Procoagulant Activity in Human Red Blood Cells

Lead (Pb) is a ubiquitously occurring environmental heavy metal which is widely used in industry and human life. Possibly due to a global industrial expansion, recent studies have revealed the prevalent human exposure to Pb and increased risk of Pb toxicity. Once ingested by human, 95% of absorbed Pb is accumulated into erythrocytes and erythrocytes are known to be a prime target for Pb toxicity. Most of the studies were however, focused on Pb2+ whereas the effects of Pb4+, another major form of Pb on erythrocytes are poorly understood yet. In this study, we investigated and compared the effects of Pb4+, Pb2+ and other heavy metals on procoagulant activation of erythrocytes, an important factor for the participation of erythrocytes in thrombotic events in an effort to address the cardiovascular toxicity of Pb4+. Freshly isolated erythrocytes from human were incubated with Pb4+, Pb2+, Cd2+ and Ag+ and the exposure of phosphatidylserine (PS), key marker for procoagulant activation was measured using flow cytometry. As a result, while Cd2+ and Ag+ did not affect PS exposure, Pb4+ and Pb2+ induced significantly PS exposure in a dose-dependent manner. Of a particular note, Pb4+ induced PS exposure with a similar potency with Pb2+. PS bearing microvesicle (MV), another important contributor to procoagulant activation was also generated by Pb4+. These PS exposure and MV generation by Pb4+ were well in line with the shape change of erythrocyte from normal discocytes to MV shedding echinocytes following Pb4+ treatment. Meanwhile, nonspecific hemolysis was not observed suggesting the specificity of Pb4+-induced PS exposure and MV generation. These results indicated that Pb4+ could induce procoagulant activation of erythrocytes through PS exposure and MV generation, suggesting that Pb4+ exposure might ultimately lead to increased thrombotic events.


INTRODUCTION
Lead (Pb) is a widely distributing environmental element and extensively used in human life for a long time. Pb exists in 4 oxidized states from +1 to +4 and of these, +2 and +4 constitutes a major portion of naturally occurring Pb. Human is exposed to Pb through industrial activities as weil as Pb contaminated food, water and Pb applied consumer products. Normal blood lead level (BLL) is below 5 fl9/dl and Pb poisoning is defined when BLL is more than 10 fl9/dl, substantially getting stricter from the guideline (> 60 fl9/dl) of 1970s (CDCP, 1997). This trend is from the recent discoveries of profound and severe toxicity of Pb to human, espe-cially to the younger children.
Once absorbed into systemic blood flow, more than 95% of Pb is associated with erythrocytes mainly in cellular membrane or hemoglobin (Goyer et al., 2001). Therefore, erythrocytes are considered as a prime target for Pb toxicity (Battistini et al., 1971). Pb denatures cellular protein and lipid components of erythrocytes (Fukumoto et al., 1983) and impairs the synthesis of hemoglobin (Monteiro et al., 1989;Waldron, 1966). In addition, a variety of toxic responses can be induced in erythrocytes by Pb such as lipid peroxidation and oxidative stress (Gurer et al., 1998;Quinlan et al., 1988), inhibition of superoxide dismutase activities and depletion of glutathione level (Ito et al., 1985;Sugawara et al., 1991). Pb exposure shortens the life span of erythrocytes and resultantly, anemia can be induced (Iavicoli et al., 2003;Waldron, 1966). It also brings about erythrocyte shape changes from discocytic normocytes to abnormal echinocytes (Kempe et al., 2005).
Erythrocytes constitute a major cell population in blood, working essentially as the oxygen carriers. Recently, it has been revealed that through the interaction with vascular endothelial cells, platelets and leukocytes, erythrocytes can actively participate in thrombosis and haemostasis (Marcus, 1990). Erythrocytes can express phosphatidylserine (PS) on the cellular membrane through the disruption of lipid asymmetry. PS exposure can be a phagocytic signal to the macrophages (Fadok et al., 1993) and more importantly, can work as a facilitator of procoagulation pathways by providing a site for the assembly of the prothrombinase and tenase complex, leading to thrombin generation and clotting (Zwaal, 1978;Zwaal et al., 1977).
Previously, we have shown that Pb 2 + induces procoagulant activation of erythrocytes through PS exposure (Shin et al., 2007). However, there was no information available about the effects of Pb 4 + on erythrocytes. In this study, we investigated and compared the effects of Pb 4 + and Pb 2 + on erythrocytes in terms of shape changes and procoagulation activation in an effort to enlighten the potential cardiovascular toxicity of Pb 4 +.
Preparation of erythrocytes. With an approval from the Ethics Committee of Health Service Center at Seoul National University, human blood was obtained from healthy male donors (18-25 years old) using a vacutainer with acid citrate dextrose (ACD) and a 21gauge needle (Becton Dickinson, Franklin Lakes, NJ) on the day of each experiment. Platelet rich plasma and buffy coat were removed by aspiration after centrifugation at 200 9 for 15 min. Packed erythrocytes were washed 3 times with phosphate buffered saline (PBS: 1 mM KH2P04, 154 mM NaCI, 3 mM Na2HP04, pH 7.4) and once with Tris buffer (TBS: 15 mM Tris-HCI, 150 mM NaCI, 5 mM KCI, 2 mM MgCI2, pH 7.4). Washed erythrocytes were resuspended in TBS to a cell concen-tration of 5 x 10 7 cells/ml and final CaCI 2 concentration was adjusted to 1 mM prior to use.
Detection of hemolysis. For detecting hemolysis, after erythrocytes were treated with vehicle (DM SO) or lead(IV) acetate for 4 hr at 37°C, the incubation was stopped by centrifugation (1 min at 12,000 g) and supernatants were harvested. Hemolysis of control erythrocytes in distilled water was defined as complete hemolysis and the extent of hemolysis was determined spectrophotometrically at 540 nm. The blank value was determined with vehicle (DMSO), respectively. Sam pie values were expressed as the percentage of complete hemolysis.
Flow cytometric analysis of phosphatidylserine exposure and microvesicle generation. Annexin V-FITC was used as a marker for phosphatidylserine (PS) detection, while anti-glycophorin A-RPE was used as an identifier of erythrocytes. Negative controls for annexin V binding were stained with annexin V-FITC in the presence of EDTA 2.5 mM instead of CaCI 2 2.5 mM. Sampies were analyzed on the flow cytometer FACScalibur™ (Becton Dickinson, San Jose, CA) equipped with argonion laser emitting at 488 nm. The light scatter and fluorescence channels were set on log scale. Data from 10,000 events were collected and analyzed using CellQuest™ Pro software.
Microscopic observation using scanning e/ectron microscopy. After fixation with 2% glutaraldehyde solution for 1 hr at 4°C, the erythrocytes were centrifuged and washed 3 times with PBS, and followed by post-fixation with 1 % osmium tetroxide for 30 min at room temperature. After washing with PBS several times, the sampies were dehydrated serially with 50, 75, 90 and 100% ethanol. After drying and coating with gold, the images were observed on scanning electron microscope (JEOL, Japan).
Statistical analysis. The means and standard errors of means were calculated for all treatment groups. The data were subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple range test or student t-test to determine which means were significantly different from the control. In all cases, a p value of < 0.05 was used to determine significant differences. sure on outer eellular membrane is an important marker for the proeoagulant aetivation of erythroeytes. PS exposure was measured using annexin V-FITC by flow eytometry analysis after erythroeytes were ineubated with Pb 4 +, Pb 2 +, Cd 2 + and Ag+ or vehicle at 37°C. As a result, PS exposure was signifieantly inereased by Pb 2 + and Pb 4 + while no effeets eould be found with Cd 2 + or Ag+ (Fig. 1) ...   Pb 4 + (j.!M) bution from nonspecific hemotoxicity was negligible (Fig. 3).

Effects
In addition to PS exposure on erythrocyte mem-  brane, PS bearing microvesicles (MV) shed from erythrocytes could contribute to the enhancement of procoagulant activity. To investigate whether Pb 4 + could induce MV generation in erythrocytes, MV was identified with annexin V-FITC and anti-glycophorin A-RPE through forward scatter in flow cytometry. Although with a less potency than PS exposure, Pb 4 + increased significantly the generation of MV from erythrocytes significantly in a concentration and time-dependent manner (Fig. 4).

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Effects of Pb 4 + on morphological change in RBCs. PS exposure and MV generation often accompany shape changes in erythrocytes. Especially, MV generation is closely related with abnormal shapes of erythrocytes like echinocytes, spherocytes or stomatocytes. To examine the changes in the shape of erythrocytes by Pb 4 +, erythrocytes were observed with scanning electron microscope following the exposure to Pb 4 +. As shown in Fig. 5, Pb 4 + treatment induced abnormal shape changes in erythrocytes from normal discocytic shapes to echinocytes, further confirming the Pb 4 + induced MV generation and PS exposure.

DISCUSSION
In the current study, we demonstrated that Pb 4 + could induce phosphatidylserine (PS) exposure and microvesicle (MV) generation in erythrocytes. Pb 4 + -induced PS exposure and MV generation were increased in a concentration and time-dependent manner and confirmed to be not related to nonspecific hemolysis. Along with PS exposure and MV generation, Pb 4 + induced abnormal shape changes from normal discocytic shapes into echinocytic shapes. These results suggest the potential roles of procoagulant activation of erythrocytes in Pb 4 +_ associated cardiovascular events.
As shown in Fig. 2 and 4, Pb 4 + induced PS exposure and MV generation potently. PS exposed on erythrocyte membrane provides a site for the assembly of tenase and prothrombinase during blood coagulation, facilitating the conversion of factor X and prothrombin to active factor Xa and thrombin (Kalafatis et al., 1994;Mann et al., 1990). The activated factor Xa and thrombin lead to the acceleration of blood coagulation, that is, procoagulant activation. Erythrocytes from many pathological states such as sickle cell anemia and thalassemia express increased procoagulant activity (Helley et al., 1996;Martin et al., 1995). In addition, endogenous thrombogenic substances like arachidonic acid, Iysophosphatidic acid and thromboxane are known to be able to induce procoagulant activation of erythrocytes Valles et al., 2002). MV generated from erythrocyte membrane could harbor PS on its outer membrane and also has a procoagulant activity (Franck et al., 1985), indicating that PS exposure and MV generation might ultimately lead to increased risk of cardiovascular diseases (Zwaal, 1978;Zwaal et al., 1977).
In this study, the procoagulant activation of erythrocytes could be induced by Pb 4 + at relatively low concentrations (5 IlM) which was similar with the active concentrations of Pb 2 + (2-5 IlM) (Fig. 1). Considering that blood lead level for lead poisoning is 10 Ilg/dl (0.5 IlM), the active concentrations of Pb 4 + are weil below clinically relevant exposure levels of lead.
Previously, it was reported that Pb can induce abnormal shape changes in erythrocytes (Baranowska-Bosiacka et al., 2003), however, there has been no information on MV generation by Pb (Shin et al., 2007). We also could not find MV generation with Pb 2 +. In the current study, we could demonstrate that Pb 4 + could induce MV generation as weil as abnormal shape changes. These results further support that Pb 4 + could be a more potent procoagulant activator of erythrocytes indicating that Pb 4 + might also be important for leadassociated cardiovascular diseases.
Previously, we have shown that PS exposure by Pb 2 + was mediated by intracellular calcium increase and ATP depletion (Shin et al., 2007). ATP depletion and calcium increase affect aminophospholipid translocases which maintain membrane lipid asymmetry. More specifically, ATP depletion and calcium increase impair the activity of flippase which recover exposed PS into inner membrane while scramblase, an enzyme scrambling lipid asymmetry is activated by calcium increase. Although further confirmatory studies are necessary, it is expected that PS exposure by Pb 4 + would also undergo ATP depletion and calcium increase. However, involvement of another novel pathway might not be excluded since Pb 4 + could induce MV generation of which aspect could not be observed with Pb 2 +.
In conclusion, this study demonstrated that micromolar concentrations of Pb 4 + could induce procoagulant activation of erythrocytes through PS exposure and PS bearing MV generations. These Pb 4 +-induced procoagulant activation of erythrocytes might lead to increased blood coagulation and thrombosis, ultimately. Along with the prevalent and global lead contamination problem, this study suggests that more efforts should be devoted into the research on Pb 4 + -induced cardiovascular toxicity.

ACKNOWLEDGMENTS
This work was supported by National Research Foun-dation of Korea Grant funded by the Korean Government (KRF-2006-E00162).