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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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Role of uremic solution on erythrocyte cytosolic Ca2+ concentration. Following exposure for 24 h to the different indicated concentration of indolic uremic solutes, erythrocytes were washed in Ringer solution and then loaded with Fluo 3/AM. Ca2+ dependent fluorescence intensity was measured with flow cytometry. The Fluo-3 mean fluorescence intensity (MFI) (arbitrary units) in erythrocytes exposed to culture media with indoxyl sulfate (IS) (A) and indoxyl-3-acetate acid (IAA) (B) was shown. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.
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toxins-07-04390-f002: Role of uremic solution on erythrocyte cytosolic Ca2+ concentration. Following exposure for 24 h to the different indicated concentration of indolic uremic solutes, erythrocytes were washed in Ringer solution and then loaded with Fluo 3/AM. Ca2+ dependent fluorescence intensity was measured with flow cytometry. The Fluo-3 mean fluorescence intensity (MFI) (arbitrary units) in erythrocytes exposed to culture media with indoxyl sulfate (IS) (A) and indoxyl-3-acetate acid (IAA) (B) was shown. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.

Mentions: To explore the mechanism responsible for the PS exposure of RBCs in indolic uremic solutes, further experiments were performed to test cytosolic Ca2+ concentration ([Ca2+]) using flow cytometry and results are represented by Ca2+ dependent fluorescence intensity. As illustrated in Figure 2A,B, in comparison to that in control conditions, median levels of IS and IAA induced a significant increase of erythrocyte cytosolic [Ca2+] after 24 h of incubation. Furthermore, there was a significant increase of [Ca2+] in maximal levels of IS (1 mM) and IAA (50 μM) than that in median uremic concentration, respectively (p < 0.001).


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)

Role of uremic solution on erythrocyte cytosolic Ca2+ concentration. Following exposure for 24 h to the different indicated concentration of indolic uremic solutes, erythrocytes were washed in Ringer solution and then loaded with Fluo 3/AM. Ca2+ dependent fluorescence intensity was measured with flow cytometry. The Fluo-3 mean fluorescence intensity (MFI) (arbitrary units) in erythrocytes exposed to culture media with indoxyl sulfate (IS) (A) and indoxyl-3-acetate acid (IAA) (B) was shown. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04390-f002: Role of uremic solution on erythrocyte cytosolic Ca2+ concentration. Following exposure for 24 h to the different indicated concentration of indolic uremic solutes, erythrocytes were washed in Ringer solution and then loaded with Fluo 3/AM. Ca2+ dependent fluorescence intensity was measured with flow cytometry. The Fluo-3 mean fluorescence intensity (MFI) (arbitrary units) in erythrocytes exposed to culture media with indoxyl sulfate (IS) (A) and indoxyl-3-acetate acid (IAA) (B) was shown. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.
Mentions: To explore the mechanism responsible for the PS exposure of RBCs in indolic uremic solutes, further experiments were performed to test cytosolic Ca2+ concentration ([Ca2+]) using flow cytometry and results are represented by Ca2+ dependent fluorescence intensity. As illustrated in Figure 2A,B, in comparison to that in control conditions, median levels of IS and IAA induced a significant increase of erythrocyte cytosolic [Ca2+] after 24 h of incubation. Furthermore, there was a significant increase of [Ca2+] in maximal levels of IS (1 mM) and IAA (50 μM) than that in median uremic concentration, respectively (p < 0.001).

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