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Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

Gao C, Ji S, Dong W, Qi Y, Song W, Cui D, Shi J - Toxins (Basel) (2015)

Bottom Line: However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear.Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS.Lactadherin acts as an efficient anticoagulant in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Technology, Harbin Medical University-Daqing, 39 Xinyang Road, Gaoxin District, Daqing 163319, China. gaochunyan1234@163.com.

ABSTRACT
Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

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Detection of phosphatidylserine (PS) exposure and microparticles (MPs) release on red blood cells (RBCs). RBCs from healthy volunteers were incubated with median and maximal uremic concentration of IS and IAA for 24 h, respectively. KCl or ethanol was utilized as their respective controls. (A,B) Lactadherin-binding percent of RBCs was evaluated by flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). (C,D) After indolic uremic solutes treatment for 24 h, MPs from 10 mL of the RBCs supernatants was harvested, and stained with Alexa Fluor 488-lactadherin and Alexa Fluor 647-CD235a. RMPs were defined as smaller than 1 μm and coexpression of lactadherin and CD235a. The number of RMP per μL culture medium was examined using flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). PS exposure and MPs release on RBCs using confocal microscopy. RBCs were stained with Alexa Fluor 488-lactadherin in the dark at room temperature. RBC membrane displayed green fluorescence when labelled by Alexa Fluor 488-lactadherin. Few lactadherin staining was observed on RBCs cultured in KCl (E) or ethanol (G). Treatment of RBCs with 1.0 mM IS (F) or 50 μM IAA (H) for 24 h led to PS externalized to the outer membrane, vesicles released from the budding of cellular membranes. Arrows indicate MP generation (appeared green) on erythrocyte membranes, and the stars indicate spherocyte. Bars represent 10 μm. PS, phosphatidylserine; IS, Indoxyl sulfate; IAA, indoxyl-3-acetate acid; RBC, red blood cell, MPs, microparticles; RMPs, RBC derived MPs.
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toxins-07-04390-f001: Detection of phosphatidylserine (PS) exposure and microparticles (MPs) release on red blood cells (RBCs). RBCs from healthy volunteers were incubated with median and maximal uremic concentration of IS and IAA for 24 h, respectively. KCl or ethanol was utilized as their respective controls. (A,B) Lactadherin-binding percent of RBCs was evaluated by flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). (C,D) After indolic uremic solutes treatment for 24 h, MPs from 10 mL of the RBCs supernatants was harvested, and stained with Alexa Fluor 488-lactadherin and Alexa Fluor 647-CD235a. RMPs were defined as smaller than 1 μm and coexpression of lactadherin and CD235a. The number of RMP per μL culture medium was examined using flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). PS exposure and MPs release on RBCs using confocal microscopy. RBCs were stained with Alexa Fluor 488-lactadherin in the dark at room temperature. RBC membrane displayed green fluorescence when labelled by Alexa Fluor 488-lactadherin. Few lactadherin staining was observed on RBCs cultured in KCl (E) or ethanol (G). Treatment of RBCs with 1.0 mM IS (F) or 50 μM IAA (H) for 24 h led to PS externalized to the outer membrane, vesicles released from the budding of cellular membranes. Arrows indicate MP generation (appeared green) on erythrocyte membranes, and the stars indicate spherocyte. Bars represent 10 μm. PS, phosphatidylserine; IS, Indoxyl sulfate; IAA, indoxyl-3-acetate acid; RBC, red blood cell, MPs, microparticles; RMPs, RBC derived MPs.

Mentions: To confirm RBC damage or eryptosis in IS and IAA, we utilized Alexa Fluor 488-lactadherin to detect PS exposure on RBCs and RMPs release by flow cytometer. After 24 h of incubation at the mean and maximal concentrations of IS and IAA found in chronic kidney disease (CKD) patients, we found that the percentage of lactadherin+ RBC in IS (0.1 mM) was significantly higher than that in control (p < 0.001), with more lactadherin+ RBC in maximal levels of IS (1 mM) than that in median levels of IS (0.1 mM) (p < 0.001). IAA also induced a significant increase in PS exposure after 24 h of incubation (p < 0.001 vs. control), and which paralleled the increasing IAA concentrations (Figure 1A,B). In another time-response experiment, IS (1 mM) or IAA (50 μM) induced a significant increase in PS exposure after 4 h of incubation, and increased rapidly during the first 24 h. From 24 h to 48 h, the proportion of lactadherin+ RBC continued to increase slowly, while a control group was almost unchanged during 48 h (Figure S1).


Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

Gao C, Ji S, Dong W, Qi Y, Song W, Cui D, Shi J - Toxins (Basel) (2015)

Detection of phosphatidylserine (PS) exposure and microparticles (MPs) release on red blood cells (RBCs). RBCs from healthy volunteers were incubated with median and maximal uremic concentration of IS and IAA for 24 h, respectively. KCl or ethanol was utilized as their respective controls. (A,B) Lactadherin-binding percent of RBCs was evaluated by flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). (C,D) After indolic uremic solutes treatment for 24 h, MPs from 10 mL of the RBCs supernatants was harvested, and stained with Alexa Fluor 488-lactadherin and Alexa Fluor 647-CD235a. RMPs were defined as smaller than 1 μm and coexpression of lactadherin and CD235a. The number of RMP per μL culture medium was examined using flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). PS exposure and MPs release on RBCs using confocal microscopy. RBCs were stained with Alexa Fluor 488-lactadherin in the dark at room temperature. RBC membrane displayed green fluorescence when labelled by Alexa Fluor 488-lactadherin. Few lactadherin staining was observed on RBCs cultured in KCl (E) or ethanol (G). Treatment of RBCs with 1.0 mM IS (F) or 50 μM IAA (H) for 24 h led to PS externalized to the outer membrane, vesicles released from the budding of cellular membranes. Arrows indicate MP generation (appeared green) on erythrocyte membranes, and the stars indicate spherocyte. Bars represent 10 μm. PS, phosphatidylserine; IS, Indoxyl sulfate; IAA, indoxyl-3-acetate acid; RBC, red blood cell, MPs, microparticles; RMPs, RBC derived MPs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663509&req=5

toxins-07-04390-f001: Detection of phosphatidylserine (PS) exposure and microparticles (MPs) release on red blood cells (RBCs). RBCs from healthy volunteers were incubated with median and maximal uremic concentration of IS and IAA for 24 h, respectively. KCl or ethanol was utilized as their respective controls. (A,B) Lactadherin-binding percent of RBCs was evaluated by flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). (C,D) After indolic uremic solutes treatment for 24 h, MPs from 10 mL of the RBCs supernatants was harvested, and stained with Alexa Fluor 488-lactadherin and Alexa Fluor 647-CD235a. RMPs were defined as smaller than 1 μm and coexpression of lactadherin and CD235a. The number of RMP per μL culture medium was examined using flow cytometry. Results represent the mean ± SD of four independent experiments (* p < 0.001). PS exposure and MPs release on RBCs using confocal microscopy. RBCs were stained with Alexa Fluor 488-lactadherin in the dark at room temperature. RBC membrane displayed green fluorescence when labelled by Alexa Fluor 488-lactadherin. Few lactadherin staining was observed on RBCs cultured in KCl (E) or ethanol (G). Treatment of RBCs with 1.0 mM IS (F) or 50 μM IAA (H) for 24 h led to PS externalized to the outer membrane, vesicles released from the budding of cellular membranes. Arrows indicate MP generation (appeared green) on erythrocyte membranes, and the stars indicate spherocyte. Bars represent 10 μm. PS, phosphatidylserine; IS, Indoxyl sulfate; IAA, indoxyl-3-acetate acid; RBC, red blood cell, MPs, microparticles; RMPs, RBC derived MPs.
Mentions: To confirm RBC damage or eryptosis in IS and IAA, we utilized Alexa Fluor 488-lactadherin to detect PS exposure on RBCs and RMPs release by flow cytometer. After 24 h of incubation at the mean and maximal concentrations of IS and IAA found in chronic kidney disease (CKD) patients, we found that the percentage of lactadherin+ RBC in IS (0.1 mM) was significantly higher than that in control (p < 0.001), with more lactadherin+ RBC in maximal levels of IS (1 mM) than that in median levels of IS (0.1 mM) (p < 0.001). IAA also induced a significant increase in PS exposure after 24 h of incubation (p < 0.001 vs. control), and which paralleled the increasing IAA concentrations (Figure 1A,B). In another time-response experiment, IS (1 mM) or IAA (50 μM) induced a significant increase in PS exposure after 4 h of incubation, and increased rapidly during the first 24 h. From 24 h to 48 h, the proportion of lactadherin+ RBC continued to increase slowly, while a control group was almost unchanged during 48 h (Figure S1).

Bottom Line: However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear.Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS.Lactadherin acts as an efficient anticoagulant in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Technology, Harbin Medical University-Daqing, 39 Xinyang Road, Gaoxin District, Daqing 163319, China. gaochunyan1234@163.com.

ABSTRACT
Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

Show MeSH
Related in: MedlinePlus