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【摘要】
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, 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.
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.
【关键词】 lysophosphatidic acid erythrocyte phosphatidylserine microvesicle thrombogenic activity
Introduction
Although erythrocytes constitute the majority of the cellular blood components, they have been commonly believed to be inert in hemostasis and thrombosis. In many clinical studies, however, it has been reported that the number of erythrocytes is closely related to bleeding time and occurrences of thrombotic diseases. 1 For example, prolonged bleeding can be treated by transfusion of erythrocytes, 2 and polycythemia vera patients, who have abnormally increased blood cell counts, often experience increased thrombosis. 3,4 In those patients, increase in erythrocyte count was the predictive marker for thrombosis.
In addition, alterations in erythrocyte membrane such as phosphatidylserine (PS) externalization and PS-bearing microvesicle generation can enable erythrocytes to participate in blood coagulation, cell adhesion, and premature erythrocyte clearance. 5 PS exposed on erythrocytes provides a site for an assembly of prothrombinase and tenase, leading to thrombin generation and clotting. 6,7 Furthermore, PS-exposing erythrocytes become more adhesive to endothelial cells, contributing to vasoocclusion. 8 PS-bearing microvesicles shed from erythrocyte membrane are also shown to have a procoagulant activity as well. 9-11 These findings strongly suggest that erythrocytes can play an active role in normal or abnormal hemostasis in certain conditions where cellular membrane perturbation occurs.
The PS exposure and/or procoagulant microvesicle generation in erythrocytes can be induced by various mechanisms such as calcium increase, 12,13 protein kinase C (PKC) activation, 14 and ATP depletion. 15 Among them, intracellular calcium increase is known as a common mechanism for both processes. The intracellular calcium levels can be increased by endogenous substances such as arachidonic acid 16 and prostaglandin (PG) E 2 17,18 in erythrocytes. Although arachidonic acid was shown to give rise to PS exposure in erythrocytes, there is, however, no report on the concomitant induction of PS exposure and procoagulant microvesicle generation or subsequent thrombotic activation by endogenous substances in erythrocytes.
Lysophosphatidic acid (LPA) is a small and structurally simple water-soluble phospholipid. Released from the activated platelets during coagulation, 19,20 LPA evokes various biological responses such as mitogenesis, 21 tumor cell invasiveness, 22 neurite retraction, 23 and mast cell development in many cell types. 24 LPA also plays an important role in pathophysiological processes like wound healing or cancer. 25,26 In particular, it is recently suggested that LPA may act as an endogenous atherogenic and thrombogenic molecule that might aggravate cardiovascular diseases like atherosclerosis, 27,28 associated in the stimulation of atherogenic processes such as vascular smooth muscle proliferation, 29 platelet aggregation, 30 vascular remodeling, 31 and fibroblast proliferation. 32
In erythrocytes, LPA can induces the influx of intracellular calcium. 33 However, it is not known whether the LPA-induced calcium influx could leads to exposure of PS or microvesicle generation, rendering erythrocytes thrombogenic enough to make contributions in hemostasis or thrombosis. In the present study, we investigated whether PS exposure and procoagulant microvesicle generation could be induced by LPA and its underlying mechanism was elucidated, in an effort to provide a new insight into the biological significances of LPA in thrombogenic activity of erythrocytes and its implications in cardiovascular diseases.
Materials and Methods
Materials
Purified human thrombin, L- -lysophosphatidic acid (LPA), glyceraldehydes, chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), phenylmethanesulfonyl fluoride (PMSF), leupeptin, sucrose, and Triton X-100 were obtained from Sigma Chemical Co. Phycoerythrin-labeled monoclonal antibody against human glycophorin A (anti-glycophorin A-RPE) was from Dako (Glostrub, Denmark) and fluo-3 acetoxymethyl ester (Fluo-3 AM) was from Molecular Probes (Eugene, Ore). Laemli sample buffer, 30% acrylamide/Bis solution, ammonium persulfate, sodium dodecyl sulfate, Tween 20, and Immune Blot polyvinylidene fluoride (PVDF) membrane were purchased from Bio-Rad Laboratories, Inc. (Hercules, Calif). Rabbit polyclonal antibody against PKC was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). 1-Palmitoyl-2-[6-[(7-nitro-2 to 1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (C 6 -NBD-PS) was purchased from Avanti Polar Lipids (Alabaster, Ala). S2238 was purchased from Chromogenix (Milano, Italy). Human recombinant tissue factor (Recombiplastin) was from Instrumentation Laboratory (Lexington, Mass). Purified human prothrombin, factor Xa, and factor Va were from Hematologic Technologies, Inc (Essex Junction, Vt). Fluorescein-isothiocyanate (FITC)-labeled annexin V (annexin V-FITC) was from Pharmingen (San Diego, Calif). Human aortic endothelial cells (HAECs) and the endothelial cell growth media (EGM) kit were purchased from Clonetics Corporation (Walkersville, Md).
Preparation of Erythrocytes and Isolation of Microvesicles
With an approval from the Ethics Committee of Health Service Center at Seoul National University, human blood was obtained from healthy male donors (18 to 25 years old) using a vacutainer with acid citrate dextrose (ACD) and a 21-gauge needle (Becton Dickinson) on the day of each experiment. Platelet-rich plasma and buffy coat were removed by aspiration after centrifugation at 200 g for 15 minutes. Packed erythrocytes were washed 3 times with phosphate buffered saline (PBS: 1 mmol/L KH 2 PO 4, 154 mmol/L NaCl, 3 mmol/L Na 2 HPO 4, pH 7.4) and once with Tris buffer (15 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L MgCl 2, pH 7.4). Washed erythrocytes were resuspended in Tris buffer to 10 8 cells/mL and final CaCl 2 concentration was adjusted to 1 mmol/L. To isolate microvesicles, erythrocytes were pelleted by centrifugation at 3000 g for 15 minutes and the supernatant containing microvesicles was collected. The microvesicles were pelleted by ultracentrifugation (Beckman Instruments Inc) at 100 000 g for 1 hour, and then the residual pellet was resuspended in Tris buffer.
Flow Cytometric Analysis of Phosphatidylserine Exposure and Microvesicle Generation
Annexin V-FITC was used as a marker for phosphatidylserine (PS) positivity, while anti-glycophorin A-RPE was for an identifier of erythrocytes. Negative controls for annexin V binding were stained with annexin V-FITC in the presence of EDTA 2.5 mmol/L instead of CaCl 2 2.5 mmol/L. Samples were analyzed on the flow cytometer, PAS (Partec) or FACStar plus (Becton Dickinson). Data from 20 000 events were collected and analyzed.
Microscopic Observation Using Scanning Electron Microscopy
After fixation with 2% glutaraldehyde solution for 1 hour at 4°C, the erythrocytes were attached onto coverslip coated with poly- l -lysine at room temperature for 30 minutes. The coverslip was rinsed with PBS 6 times and post-fixed with 1% osmium tetroxide for 30 minutes. After washing with PBS 6 times, the sample was dehydrated serially with 10, 30, 50, 70, 95, and 100% ethanol. After critical point drying and coating with platinum, the images were observed on scanning electron microscope (JEOL, Japan).
Measurement of Cytosolic Calcium in Erythrocytes
Isolated erythrocytes were diluted to a final concentration of 2 x 10 7 cells/mL and loaded with Fluo-3 AM by incubation with 3 µmol/L Fluo-3 AM for 1 hour. Subsequently, the cells were washed and then in Tris buffer to a final concentration of 10 7 cells/mL. To quench off extracellular calcium, 3 mmol/L of EGTA was added to the erythrocyte suspension. After Fluo-3 loaded erythrocytes were incubated with PBS or LPA for 15 minutes, the fluorescence was measured on flow cytometer.
Western Blot Analysis of Protein Kinase C Translocation
After treatment with LPA 20 µmol/L, erythrocytes were washed in Tris buffer at 4°C. Packed cells were suspended in cold lysis buffer (10 mmol/L HEPES, 0.25 mol/L sucrose, 0.1 mmol/L PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 5 mmol/L EDTA, pH 7.5) and sonicated for 60 seconds with Ultrasonic Processor (GEX 400, Sonics and Materials Inc) on ice followed by centrifugation at 100 000 g for 1 hour at 4°C. Upper-half supernatant was collected as a cytosolic fraction, and the pellets were resuspended in the cold lysis buffer containing 1% Triton X-100. Samples were sonicated for 30 seconds and centrifuged again. The supernatant was collected as a membrane fraction. A quantity of 100 µg protein of cytosolic fraction and 30 to 40 µg protein of membrane fraction were separated on 8% SDS-PAGE and transferred onto PVDF membrane. The membrane was incubated overnight with PKC antibody at 4°C and washed in TBS-T (20 mmol/L Tris-base, 137 mmol/L NaCl, 0.1% Tween 20, pH 7.6). After incubation with secondary antibody, immunoreactive bands were visualized using the enhanced chemiluminescence detection system. Data were quantified by densitometry after scanning, using the TINA software (Raytest, Germany).
Measurement of Flippase Activity
Flippase activity was determined based on the methods described by Bucki et al. 34 Please see supplemental materials (available online at http://atvb.ahajournals.org).
Prothrombinase Assay
Please see supplemental materials.
Measurement of Thrombin Generation in Plasma
Thrombin generation in plasma was measured according to the methods previously described by Peyrou et al. 35 Tris buffer or erythrocytes were added to plasma and the mixture was prewarmed for 2 minutes at 37°C. Under gentle magnetic stirring, thrombin formation was initiated by adding Recombiplastin diluted (1:3200) in Tris buffer containing 100 mmol/L CaCl 2. The aliquots were collected at indicated time and transferred to a Tris buffer containing 20 mmol/L EDTA. Thrombin activity was determined using the chromogenic substrate S2238.
Adherence of Erythrocytes to Human Aortic Endothelial Cells
The HAECs (6 to 8 passages) were maintained in the EGM kit at 37°C in a 95% air/5% CO 2 incubator. Before the experiments, 4 x 10 4 cells were seeded into 12-well plates and grown for 48 hours. Erythrocyte adherence to HAECs was measured using modification of the methods described by Manodori et al. 36 PBS or LPA-treated erythrocytes were washed twice and resuspended in Hanks balanced salt solution (HBSS) to a cell concentration of 2.5 x 10 7 cells/mL. After HAECs were washed twice with HBSS to remove media, the erythrocytes were layered onto confluent HAEC monolayers and incubated for 30 minutes at 37°C. After the incubation, the wells were filled completely with HBSS, sealed with parafilm, and inverted for 20 minutes at 37°C. The wells were rinsed twice with PBS to remove nonadherent erythrocytes and fixed with 2% glyceraldehydes. The number of adherent erythrocyte was counted on light microscope. The experiments were performed in triplicate and 8 fields were selected randomly to count erythrocyte number. Erythrocyte adherence was expressed as the percent increase over control.
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 multiple range test to determine which means were significantly different from the control. In all cases, a probability value of <0.05 was used to determine significance.
Results
To investigate whether LPA induces phosphatidylserine (PS) exposure on erythrocyte surface and microvesicle generation, 10 µmol/L of LPA was added to erythrocyte suspension for 15 minutes and then flow cytometric analysis was conducted. Microvesicles derived from erythrocyte membrane were identified with anti-glycophorin A antibody and the extent of PS exposure was analyzed using annexin V binding measurement. Treatment of LPA caused the release of erythrocyte membrane-derived small particles and increased the number of erythrocytes binding annexin V over control level ( Figure 1 A). The particles from erythrocytes had diameters less than 1 µm when compared with the size of 1-µm-diameter standard beads in the histogram (data not shown). This size of the particles corresponded to the microparticles reported in the previous studies. 37 Most of microvesicles (98%) also expressed PS on their surfaces. Both PS exposure and microvesicle generation were increased by LPA in a concentration-dependent manner ( Figure 1B and 1 C).
Figure 1. PS exposure and PS-bearing microvesicle (MV) generation by LPA in human erythrocytes. After human erythrocyte suspension was incubated with PBS (control) or LPA for 15 minutes at 37°C, the flow cytometric analysis was employed: with representative dot plots and histograms of erythrocyte suspension treated with PBS or 10 µmol/L of LPA (A), percentage of cells expressing PS (B), and the amount of microvesicle released from erythrocytes by various concentrations of LPA (C) were shown. Values are mean±SEM of 4 to 5 independent experiments from different blood donors. *Significant differences from control group ( P <0.05).
Shape change precedes the microvesicle release in erythrocytes. Under normal condition, erythrocytes show discocytic shape. When some stimuli are applied to the erythrocytes, they transform to echinocytic shape, in which spiculated membrane can be shed from the cell as a form of microvesicle. When the echinocytes undergo further exovesiculation, cell surface area is lost leading to an increased sphericity of erythrocytes. Accordingly, we observed the morphological changes using scanning electron microscopy to confirm the microvesicle generation by LPA treatment. Echinocytes were found in LPA-treated groups, whereas all the cells in control group showed normal discocytic shape ( Figure 2 ). When the higher concentration of LPA (20 µmol/L) was added to the cells, spherocytes as well as echinocytes were observed ( Figure 2 C). This observation is consistent with the previous result ( Figure 1 ) that microvesicles were produced by LPA treatment.
Figure 2. LPA-induced shape changes in human erythrocytes. Erythrocytes were incubated with PBS (A) or 10 µmol/L (B) or 20 µmol/L (C) LPA for 15 minutes at 37°C. The cells were fixed and the morphological changes were examined using scanning electron microscope. Representative data of 3 independent experiments from different blood donors was shown here. (Original magnification: x 3500).
Because calcium influx into erythrocytes is known to be a common mechanism of PS exposure and microvesiculation, the role of calcium in the LPA-induced events was investigated. As shown in Figure 3 A, LPA stimulated calcium influx in erythrocytes in consistent with the previous report. 33 When extracellular calcium was chelated with the excess of EGTA (3 mmol/L) addition, the elevation in intracellular calcium level by LPA was abolished completely ( Figure 3 A). However, neither PS exposure nor microvesicle generation caused by LPA was affected by the calcium chelator ( Figure 3 B). These results imply that intracellular calcium increase is not a key mechanism for the PS exposure and microvesicle generation induced by LPA in erythrocytes.
Figure 3. Calcium dependency on LPA-induced PS exposure and microvesicle generation. A, Erythrocytes loaded with fluo-3 were treated with PBS (control) or 10 µmol/L LPA in the absence or presence of 3 mmol/L EGTA. After 15 minutes incubation at 37°C, intracellular calcium levels were evaluated by fluorescent cells using flow cytometer. B, The extent of PS exposure and the microvesicle generation were also measured by flow cytometry. Open and closed bars show the results in the absence and presence of EGTA, respectively. Values are mean±SEM of 3 independent experiments from different blood donors.
In addition to calcium level increase, activation of PKC could induce phospholipid scrambling, leading to PS exposure on erythrocytes. 14 To date, PKC, a calcium-dependent isoform and PKC, a calcium-independent isoform have been reported to be expressed in erythrocytes. 38 To determine whether PKC is involved in the scrambling of the membrane phospholipid by LPA, inhibitors of PKC, chelerythrine, and H-7, were used. Before LPA addition, erythrocytes were pretreated with chelerythrine or H-7 for 5 minutes. Both chelerythrine and H-7 decreased LPA-induced PS exposure by 58% and 42%, respectively ( Figure 4 A). In addition, the PKC inhibitors also suppressed microvesicle generation in part. Because calcium dependency could not be found, we investigated whether PKC, a calcium-independent PKC isoform in erythrocytes, was activated by LPA. Activation of PKC was confirmed by the Western blot analysis of translocation from cytosol to membrane ( Figure 4 B), suggesting that calcium-independent PKC activation is involved in PS exposure and microvesicle generation induced by LPA in erythrocytes. In addition, we examined the effect of LPA on the activity of flippase, which is important in the maintenance of membrane asymmetry by transporting PS from outer to inner leaflet of plasma membrane against concentration gradient. 39 The inward transport of NBD-PS, the fluorescent probe for flippase, was significantly decreased in the presence of LPA, indicating that flippase activity was inhibited by LPA treatment (supplemental Figure I).
Figure 4. Role of PKC in PS exposure and microvesicle generation induced by LPA. A, The PS exposure and the microvesicle generation induced by 20 µmol/L LPA were measured after pretreatment with PKC inhibitors, 50 µmol/L chelerythrine (CHEL) or 200 µmol/L H-7, for 5 minutes. B, Erythrocytes were treated with 20 µmol/L LPA for various time periods and lysed for Western blot detection of PKC. Representative data were shown and the time course of PKC translocation was plotted. Values are mean±SEM of 3 to 5 independent experiments from different blood donors. *Significant differences from control group; significant differences from LPA alone group ( P <0.05).
To investigate whether PS exposure induced by LPA is strong enough to elicit a thrombogenic activity in erythrocytes, LPA-induced procoagulant activity and erythrocyte adherence to HAECs were determined. The remnant erythrocytes and microvesicles isolated from LPA-treated erythrocyte suspension increased thrombin generation significantly, as determined by prothrombinase assay (supplemental Figure II). Furthermore, the addition of remnant erythrocytes and microvesicles to plasma enhanced the coagulation process initiated by recombinant human tissue factor ( Figure 5 A), suggesting that LPA-exposed erythrocytes could contribute to coagulation process through PS-exposure and microvesicle generation. Another implication of PS-exposure on erythrocytes is to promote adhesion to vascular endothelial cells. 8,36 Treatment with LPA significantly enhanced the adhesion of erythrocytes to HAECs, suggesting that LPA-stimulated erythrocytes could be more thrombogenic than normal erythrocytes ( Figure 5 B).
Figure 5. Enhancement of thrombin generation and erythrocyte adherence by LPA treatment. A, Erythrocyte suspension was incubated with PBS or 10 µmol/L of LPA for 15 minutes and remnant erythrocytes were isolated from the suspension. Microvesicles were isolated from the LPA (20 µmol/L)-treated erythrocyte suspension. Thrombin generation in plasma was initiated by Recombiplastin and calcium. B, LPA-treated erythrocytes were incubated with HAECs for 30 minutes and further incubated for 20 minutes at inverted state. After washing and fixation, adhered erythrocytes to HAECs were counted on microscope. Values are mean±SEM of 3 to 6 independent experiments from different blood donors. *Significant differences from control group ( P <0.05).
Discussion
In the current investigation, LPA was found to induce PS exposure and procoagulant microvesicle generation in human erythrocytes. The microvesicle generation was further confirmed by microscopic observation of morphological changes. Calcium-independent PKC activation was involved in these processes, although any direct relationship with calcium influx was not found. Consistent with the PS exposure, LPA treatment to erythrocytes inhibited flippase activity. Furthermore, the microvesicles and remnant cells isolated from LPA-treated erythrocytes actually potentiated prothrombinase activity and thrombin generation in plasma. Besides, LPA-exposed erythrocytes showed increased adherence to HAECs, suggesting LPA could induce clinically significant thrombus formation. Above all, these results were observed at physiologically relevant concentrations of LPA. LPA concentration in coagulated serum generally ranges from 1 to 10 µmol/L and can reach as high as 20 µmol/L. 40,41 Higher concentration up to 200 µmol/L has also been found in plasma and malignant ascites fluid of ovarian and cervical cancer patients. 42,43 The concentration of LPA used in this study did not exceed 20 µmol/L. Especially, the adhesion of erythrocytes to HAECs was observed even at as low as 5 µmol/L ( Figure 5 B), suggesting that the thrombogenic activity observed in this study may appear in vivo as well.
It is suggested that LPA may act as an endogenous atherogenic and thrombogenic molecule in platelet, smooth muscle, and fibroblast that might aggravate cardiovascular diseases such as atherosclerosis. 27,28 However, study on atherogenic and thrombogenic effect of LPA in erythrocytes has been very limited. Together with the multiple effects on platelets, smooth muscle, and fibroblast, thrombogenic effects of LPA on erythrocytes shown in this study may play a role in the precipitation of thrombosis and cardiovascular diseases induced by LPA in susceptible population such as cancer and atherosclerosis patients.
Other endogenous substances like arachidonic acid and thromboxane A 2 mimic, could also express PS on erythrocyte surface, 16 but the extent of PS exposure by the substances was minimal, less than 5% of total erythrocyte population. However, the results in Figure 1A and 1 B revealed that LPA increased PS-exposing cells up to 40%, suggesting that LPA might be one of the most potent endogenous agonists that induce PS exposure in human erythrocytes. In addition to PS exposure, LPA induced concomitant generation of microvesicles from erythrocytes ( Figure 1A and 1 C). So far, most studies on microvesicle generation have been focused on genetically defected erythrocytes from patients such as sickle cell anemia 44 or normal erythrocytes challenged with exogenous substance such as calcium ionophore. 45,46 This is the first report on the procoagulant microvesicle generation by an endogenous substance in normal erythrocytes. The generation of procoagulant microvesicles combined with PS exposure would render LPA to be more thrombogenic in human erythrocytes.
The PS exposure on cell membrane and procoagulant microvesicle formation can lead to the enhancement of blood coagulation and cell adherence. Although LPA induced the PS exposure and PS-exposing microvesicles in erythrocytes, it was not certain that these alterations would be sufficient enough to have biological significances. Therefore, procoagulant activities of LPA-exposed erythrocytes and isolated microvesicles were evaluated using thrombin generation in plasma as a marker. As expected, it was ascertained that erythrocytes exposed to LPA and microvesicles generated by LPA enhance coagulation process. In addition, erythrocyte adherence to endothelial cells was significantly increased by LPA treatment as well. These results indicate that the PS exposure and microvesicle generation induced by LPA have biological significances which may contribute to cardiovascular diseases.
It is generally known that PS exposure and microvesicle generation are mediated by intracellular calcium increase. 45,47 The calcium dependency in LPA-induced PS exposure, however, was not found in this study. Consistent with our results, de Jong et al could not find a clear correlation between elevated calcium levels and PS exposure in erythrocytes. 14 Instead, calcium-independent PKC activation is involved in the LPA-induced events as shown by PKC inhibitors and Western blot analysis of PKC translocation induced by LPA ( Figure 4 ). Supporting these results, Seewald et al reported that LPA can activate various subtypes of PKC including calcium independent type PKC via receptor in vascular smooth muscle cells, 48 and PKC is known to be the representative calcium-independent PKC in erythrocytes. 38 PKC activation can influence cytoskeletal integrity and erythrocyte functions by phosphorylation of membrane protein like band 4.1, 4.9, and adducin, 49-51 which could mediate the LPA-induced changes in erythrocytes.
Effects of LPA have been reported to be mediated by specific G protein-coupled receptor. 25 However, suramine, a G protein uncoupler, did not block either PS exposure or microvesicle generation induced by LPA in erythrocytes (data not shown), suggesting that these effects are independent of G protein-coupled receptor-mediated pathway. These data agree with the previous report 52 that LPA released from activated platelets can diffuse directly into other neighboring cells, activating intracellular signaling pathways.
In summary, we demonstrated that human erythrocytes respond to endogenous LPA to express PS on their surface and release procoagulant microvesicles, acquiring a thrombogenic activity ( Figure 6 ) mediated through calcium independent PKC activation. These effects were seen at levels of LPA comparable to that observed in some patients. Our study will provide new insights into the role of LPA and erythrocytes in the development of cardiovascular diseases.
Figure 6. Schematic diagram for LPA-induced thrombogenic activity in human erythrocytes. Erythrocytes respond to endogenous LPA to express PS on their surface and release procoagulant microvesicles. The microvesicles and remnant cells formed from LPA-treated erythrocytes can induce thrombin generation in plasma and enhance the adhesion of erythrocytes to ECs, acquiring a thrombogenic activity.
Acknowledgments
Sources of Funding
This work was supported by the NITR project from Korea Food Drug Administration (KFDA). The authors also thank Health Fellowship Foundation for the financial support.
Disclosures
None.
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作者单位：College of Pharmacy (S.-M.C., O.-N.B., K.-M.L., J.-Y.N., M.-Y.L., J.-H.C.), Seoul National University, Seoul, and the College of Medicine (Y.-S.J.), Ajou University, Suwon, Korea.