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Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

ABSTRACT: The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis.

SUBMITTER: Gao C

PROVIDER: S-EPMC4646287 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

Gao Chunyan C Xie Rui R Yu Chengyuan C Ma Ruishuang R Dong Weijun W Meng Huan H Zhang Yan Y Si Yu Y Zhang Zhuo Z Novakovic Valerie V Zhang Yong Y Kou Junjie J Bi Yayan Y Li Baoxin B Xie Rujuan R Gilbert Gary E GE Zhou Jin J Shi Jialan J

PloS one 20151116 11

The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrom ...[more]

PMID: 26580207

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Project description:Annexin-V (PAP-I, lipocortin-V) acts as a potent anticoagulant in vitro by binding to negatively charged phospholipids with higher affinity than vitamin K-dependent proteins, with a Kd in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic potential of phospholipids in pro- and anti-coagulant reactions in purified systems and at the surface of endothelial cells in culture after stimulation. Procoagulant tissue factor and anticoagulant thrombomodulin activities were compared by using specific two-stage amidolytic assays performed with purified proteins. Procoagulant activity was estimated by the generation of Factor Xa by the Factor VII(a)-tissue factor complex. Anticoagulant activity was estimated by the generation of activated protein C by either the thrombin-thrombomodulin complex or Factor Xa. Annexin-V induced a decrease of 70% of thrombomodulin activity when thrombomodulin (5.4-214 nM) was reconstituted into phosphatidylcholine/phosphatidylserine (1:1, mol/mol) vesicles at 37.5 or 75 microM-phospholipid concentration, the apparent Ki being 0.5 microM at 75 microM-lipid. The saturating concentration of annexin-V was dependent on phospholipid concentration, but was independent of the phospholipid/thrombomodulin ratio. By contrast, when thrombomodulin was not reconstituted in vesicles, annexin-V had no effect. At 2 microM, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75 microM-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1 microM, annexin-V totally inhibited tissue-factor activity present in crude brain thromboplastin. In the absence of stimulation, human endothelial cells in culture expressed significant thrombomodulin activity and no detectable tissue-factor activity. Basal thrombomodulin activity was only slightly inhibited (less than 15%) by 0.5 microM-annexin-V. Phorbol myristate acetate (PMA) induced the expression of tissue-factor activity and decreased thrombomodulin activity at the endothelial-cell surface. Annexin-V, at a concentration of 16 microM, caused an 80% decrease of tissue-factor activity induced by PMA at 10 ng/ml, whereas it inhibited thrombomodulin activity by only 15% on the same stimulated cells. Our results confirm that annexin-V inhibits, in vitro, procoagulant tissue-factor activity and anticoagulant activities (activation of protein C by the thrombin-thrombomodulin complex and by Factor Xa), through phospholipid-dependent mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Dataset Information

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

Publications

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

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