Department of Nephrology, the First Hospital, Harbin, China.
Department of Hematology, the First Hospital, Harbin, China.
Nephrol Dial Transplant. 2018 Dec 1;33(12):2115-2127. doi: 10.1093/ndt/gfy027.
Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with diabetic kidney disease (DKD). This study was designed to evaluate whether exposed PS on microparticles (MPs) and MP-origin cells were involved in the hypercoagulability in DKD patients.
DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate, defined as normoalbuminuria (No-A) (<30 mg/24 h), microalbuminuria (Mi-A) (30-299 mg/24 h) or macroalbuminuria (Ma-A) (>300 mg/24 h), and compared with healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells (BCs) and human umbilical vein endothelial cells (HUVECs) were treated with 25, 5 or 2.5 mmol/L glucose as well as 3-12 mg/dL uric acid and cells were evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure and fibrin strands were observed using confocal microscopy.
Using flow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and BCs were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with Ma-A was significantly higher than in patients with No-A. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with Ma-A versus serum from patients with Mi-A or normoalbuminuria. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated colocalization of fibrin with PS on HUVECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by ∼80%. In vitro, BCs and endothelial cells exposed more PS in hypoglycemia or hyperglycemia. Interestingly, reconstitution experiments showed that hypoglycemia-treated cells could be further activated or injured when recovery is obtained reaching hyperglycemia. Moreover, uric acid induced PS exposure on cells (excluding platelets) at concentrations >6 mg/dL. Linear regression analysis showed that levels of PS+ BCs and microparticles were positively correlated with uric acid and proteinuria, but negatively correlated with glomerular filtration rate.
Our results suggest that PS+ MPs and MP-origin cells play procoagulant roles in patients with DKD. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.
关于磷脂酰丝氨酸（PS）在糖尿病肾病（DKD）患者促凝活性（PCA）中的作用，人们知之甚少。本研究旨在评估暴露于微粒（MPs）和 MP 来源细胞上的 PS 是否参与了 DKD 患者的高凝状态。
根据尿白蛋白排泄率将 DKD 患者（n=90）分为三组，定义为正常白蛋白尿（No-A）（<30mg/24h）、微量白蛋白尿（Mi-A）（30-299mg/24h）或大量白蛋白尿（Ma-A）（>300mg/24h），并与健康对照组（n=30）进行比较。使用乳酰脱氢酶来定量 MPs 及其原始细胞上的 PS 暴露情况。将健康血细胞（BCs）和人脐静脉内皮细胞（HUVECs）用 25、5 或 2.5mmol/L 葡萄糖以及 3-12mg/dL 尿酸处理，并通过凝血时间和纯化的凝血复合物测定来评估细胞。通过浊度法测定纤维蛋白的产生。使用共聚焦显微镜观察 PS 暴露和纤维丝。
使用流式细胞术，我们发现与对照组相比，患者的 PS+ MPs（来自血小板、红细胞、HUVECs、中性粒细胞、单核细胞和淋巴细胞）和 BCs 明显更高。此外，Ma-A 患者的 PS+ MPs 和 BCs 数量明显高于 No-A 患者。同样，我们观察到与 Mi-A 或正常白蛋白尿患者的血清相比，来自 Ma-A 患者的血清培养的 HUVECs 上 PS 暴露明显增加。此外，循环 PS+ MPs 与 PS+细胞共同作用，导致每个 DKD 组的凝血时间明显缩短，FXa/凝血酶生成和纤维蛋白形成显著增加。共聚焦显微镜图像显示纤维蛋白与 HUVECs 上的 PS 共定位。此外，用乳酰脱氢酶阻断 MPs 和细胞上的暴露 PS 可抑制 PCA 约 80%。在体外，低血糖或高血糖会使 BCs 和内皮细胞暴露更多的 PS。有趣的是，再灌注实验表明，当血糖恢复到高血糖水平时，经过低血糖处理的细胞可能会进一步被激活或受损。此外，尿酸在浓度>6mg/dL 时会诱导细胞（不包括血小板）上的 PS 暴露。线性回归分析表明，PS+BCs 和微颗粒的水平与尿酸和蛋白尿呈正相关，与肾小球滤过率呈负相关。
我们的研究结果表明，PS+ MPs 和 MP 来源细胞在 DKD 患者中发挥促凝作用。阻断 PS 可能成为预防这些患者血栓形成的一种新的治疗方法。
