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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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Recalcification time and inhibition assay. RBCs were treated with different concentrations of IS or IAA for 24 h, cells and RMPs were collected, respectively. KCl or ethanol was utilized as their respective controls. Coagulation times of 100 μL RBCs (1 × 108) and RMPs (prepared from 10 mL of the RBCs supernatants) in each group of IS (A) and IAA (B) are shown. PCA of RBCs and RMPs that incubated in IS (C) and IAA (D) were detected in the absence or presence of 128 nM lactadherin. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.
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toxins-07-04390-f004: Recalcification time and inhibition assay. RBCs were treated with different concentrations of IS or IAA for 24 h, cells and RMPs were collected, respectively. KCl or ethanol was utilized as their respective controls. Coagulation times of 100 μL RBCs (1 × 108) and RMPs (prepared from 10 mL of the RBCs supernatants) in each group of IS (A) and IAA (B) are shown. PCA of RBCs and RMPs that incubated in IS (C) and IAA (D) were detected in the absence or presence of 128 nM lactadherin. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.

Mentions: Further experiments explored whether indolic uremic solutes triggered PCA of RBCs. We first evaluated the PCA of RBCs and RMPs by recalcification-time assays in the absence or presence of IS and IAA. Incremental PCA was exhibited by reductive clotting time. As shown in Figure 4A,B, the coagulation time was significantly reduced in IS (0.1 mM) and IAA (20 μM) compared with RBCs in control medium (KCl for IS, ethanol for IAA) after 24 h of incubation (p < 0.001), with shorter coagulation time in maximal levels of IS (1 mM) and IAA (50 μM) than in median uremic concentration (p < 0.001). For RMPs, IS and IAA enhanced the PCA also in a concentration-dependent fashion. The reduction of coagulation time was in accord with the number of RMPs. In order to explore the relationship between PS exposure and PCA of RBCs that induced by IS or IAA, we performed coagulation inhibition assays. PCA of both RBCs and RMPs were almost completely inhibited by 128 nM lactadherin, as described in Figure 4C,D.


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)

Recalcification time and inhibition assay. RBCs were treated with different concentrations of IS or IAA for 24 h, cells and RMPs were collected, respectively. KCl or ethanol was utilized as their respective controls. Coagulation times of 100 μL RBCs (1 × 108) and RMPs (prepared from 10 mL of the RBCs supernatants) in each group of IS (A) and IAA (B) are shown. PCA of RBCs and RMPs that incubated in IS (C) and IAA (D) were detected in the absence or presence of 128 nM lactadherin. 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-f004: Recalcification time and inhibition assay. RBCs were treated with different concentrations of IS or IAA for 24 h, cells and RMPs were collected, respectively. KCl or ethanol was utilized as their respective controls. Coagulation times of 100 μL RBCs (1 × 108) and RMPs (prepared from 10 mL of the RBCs supernatants) in each group of IS (A) and IAA (B) are shown. PCA of RBCs and RMPs that incubated in IS (C) and IAA (D) were detected in the absence or presence of 128 nM lactadherin. Data are displayed as mean ± SD for triplicate samples of independent experiments, * indicate p < 0.001.
Mentions: Further experiments explored whether indolic uremic solutes triggered PCA of RBCs. We first evaluated the PCA of RBCs and RMPs by recalcification-time assays in the absence or presence of IS and IAA. Incremental PCA was exhibited by reductive clotting time. As shown in Figure 4A,B, the coagulation time was significantly reduced in IS (0.1 mM) and IAA (20 μM) compared with RBCs in control medium (KCl for IS, ethanol for IAA) after 24 h of incubation (p < 0.001), with shorter coagulation time in maximal levels of IS (1 mM) and IAA (50 μM) than in median uremic concentration (p < 0.001). For RMPs, IS and IAA enhanced the PCA also in a concentration-dependent fashion. The reduction of coagulation time was in accord with the number of RMPs. In order to explore the relationship between PS exposure and PCA of RBCs that induced by IS or IAA, we performed coagulation inhibition assays. PCA of both RBCs and RMPs were almost completely inhibited by 128 nM lactadherin, as described in Figure 4C,D.

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