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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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Formation and inhibition assays of procoagulant enzyme complexes. After exposure for 24 h to the different indicated concentration of IS or IAA or KCl (control) or ethanol (control), erythrocytes were washed with Ringer solution. Intrinsic FXa formation was measured in the presence of FIXa, FVIII and thrombin. Thrombin generation was investigated in the presence of FXa and FVa. Intrinsic FXa and thrombin production of 105 RBCs in each group of IS (A) and IAA (B) are shown. The capacity of 128 nM lactadherin to block procoagulant enzyme complexes on RBCs that incubated in IS (C) and IAA (D) was evaluated. Results represent the mean ± SD for triplicate of independent experiments, * indicate p < 0.001.
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toxins-07-04390-f005: Formation and inhibition assays of procoagulant enzyme complexes. After exposure for 24 h to the different indicated concentration of IS or IAA or KCl (control) or ethanol (control), erythrocytes were washed with Ringer solution. Intrinsic FXa formation was measured in the presence of FIXa, FVIII and thrombin. Thrombin generation was investigated in the presence of FXa and FVa. Intrinsic FXa and thrombin production of 105 RBCs in each group of IS (A) and IAA (B) are shown. The capacity of 128 nM lactadherin to block procoagulant enzyme complexes on RBCs that incubated in IS (C) and IAA (D) was evaluated. Results represent the mean ± SD for triplicate of independent experiments, * indicate p < 0.001.

Mentions: We further investigated the capacity of RBCs to support intrinsic FXa and thrombin that contribute to PCA. As depicted in Figure 5A, the production of the two procoagulant enzyme complexes was increased in median uremic concentration of IS (0.1 mM) compared with controls (p < 0.001), and more higher in maximal levels of IS (1 mM). Similar results were obtained on IAA treated RBC, the rise of thrombin and intrinsic FXa production paralleled the increasing IAA concentration (Figure 5B). Inhibition assays of FXa and prothrombinase productions were also performed. Over 90% of the production of two procoagulant enzyme complexes was inhibited by 128 nM lactadherin (Figure 5C,D). Results from inhibition assays confirmed further that PS played a crucial role in the PCA of indolic uremic solutes treated RBCs.


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)

Formation and inhibition assays of procoagulant enzyme complexes. After exposure for 24 h to the different indicated concentration of IS or IAA or KCl (control) or ethanol (control), erythrocytes were washed with Ringer solution. Intrinsic FXa formation was measured in the presence of FIXa, FVIII and thrombin. Thrombin generation was investigated in the presence of FXa and FVa. Intrinsic FXa and thrombin production of 105 RBCs in each group of IS (A) and IAA (B) are shown. The capacity of 128 nM lactadherin to block procoagulant enzyme complexes on RBCs that incubated in IS (C) and IAA (D) was evaluated. Results represent the mean ± SD for triplicate 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-f005: Formation and inhibition assays of procoagulant enzyme complexes. After exposure for 24 h to the different indicated concentration of IS or IAA or KCl (control) or ethanol (control), erythrocytes were washed with Ringer solution. Intrinsic FXa formation was measured in the presence of FIXa, FVIII and thrombin. Thrombin generation was investigated in the presence of FXa and FVa. Intrinsic FXa and thrombin production of 105 RBCs in each group of IS (A) and IAA (B) are shown. The capacity of 128 nM lactadherin to block procoagulant enzyme complexes on RBCs that incubated in IS (C) and IAA (D) was evaluated. Results represent the mean ± SD for triplicate of independent experiments, * indicate p < 0.001.
Mentions: We further investigated the capacity of RBCs to support intrinsic FXa and thrombin that contribute to PCA. As depicted in Figure 5A, the production of the two procoagulant enzyme complexes was increased in median uremic concentration of IS (0.1 mM) compared with controls (p < 0.001), and more higher in maximal levels of IS (1 mM). Similar results were obtained on IAA treated RBC, the rise of thrombin and intrinsic FXa production paralleled the increasing IAA concentration (Figure 5B). Inhibition assays of FXa and prothrombinase productions were also performed. Over 90% of the production of two procoagulant enzyme complexes was inhibited by 128 nM lactadherin (Figure 5C,D). Results from inhibition assays confirmed further that PS played a crucial role in the PCA of indolic uremic solutes treated RBCs.

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