Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1
Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1
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DOI：
10.1016/j.jtcvs.2005.08.050
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发表时间：
2006-01-01
影响因子：
6
通讯作者：
Landis, RC
中科院分区：
文献类型：
--
作者：
Day, JRS;Taylor, KM;Landis, RC
目的：凝血酶在体外循环过程中大量产生，通过激活高亲和力的凝血酶受体蛋白激活受体1介导血小板聚集和炎症反应等不良反应，该受体表达于血小板和内皮细胞上。因此，蛋白酶激活受体1的拮抗作用可能具有广泛的治疗意义。抑肽酶在临床上用于减少输血需求和体外循环的炎症反应，已被证明在体外和体内都能抑制血小板上的蛋白水解酶激活的受体1。方法：在体外培养的人脐静脉内皮细胞中，加入抑肽酶(200-1600个激肽释放酶抑制单位/毫升)或加入0.02-0.15U/mL的α-凝血酶，观察其对血管内皮细胞的表达和功能的影响。使用仅检测未激活受体的抗体SPAN-12和凝血酶介导的钙离子通量来评估蛋白酶激活的受体1的激活情况。其他被研究的凝血酶依赖的炎症途径包括p42/44丝裂原活化蛋白激酶的磷酸化，早期生长反应1转录因子的上调，以及促炎细胞因子白细胞介素6的产生。结果：抑肽酶能显著减少培养的内皮细胞蛋白水解酶激活的受体1受体的断裂(P<；.0001)和凝血酶引起的钙离子通量的减少。抑肽酶通过p42/44丝裂原活化蛋白激酶(P<；.05)和早期生长反应1转录因子(P<；.05)抑制细胞内信号转导，并抑制凝血酶诱导的白介素6的分泌(P<；.005)。结论：抑肽酶可通过阻断蛋白酶激活受体1而抑制血管内皮细胞的凝血酶激活及其下游炎症反应。本研究结果为临床报道抑肽酶的抗炎作用提供了新的分子基础。
Objective: Thrombin is generated in significant quantities during cardiopulmonary bypass and mediates adverse events, such as platelet aggregation and proinflammatory responses, through activation of the high-affinity thrombin receptor protease-activated receptor 1, which is expressed on platelets and endothelium. Thus antagonism of protease-activated receptor 1 might have broad therapeutic significance. Aprotinin, used clinically to reduce transfusion requirements and the inflammatory response to bypass, has been shown to inhibit protease-activated receptor 1 on platelets in vitro and in vivo. Here we have examined whether aprotinin inhibits endothelial protease-activated receptor 1 activation and resulting proinflammatory responses induced by thrombin.Methods: Protease-activated receptor 1 expression and function were examined ill cultured human umbilical vein endothelial cells after treatment with a-thrombin at 0.02 to 0.15 U/mL in the presence or absence of aprotinin (200-1600 kallikrein inhibitory units/mL). Protease-activated receptor 1 activation was assessed by using an antibody, SPAN-12, which detects only the unactivated receptor, and thrombin-mediated calcium fluxes. Other thrombin-dependent inflammatory pathways investigated were phosphorylation of the p42/44 mitogen-activated protein kinase, upregulation of the early growth response 1 transcription factor, and production of the proinflammatory cytokine interleukin 6.Results: Pretreatment of cultured endothelial cells with aprotinin significantly spared protease-activated receptor 1 receptor cleavage (P < .0001) and abrogated calcium fluxes caused by thrombin. Aprotinin inhibited intracellular signaling through p42/44 mitogen-activated protein kinase (P < .05) and early growth response 1 transcription factor (P < .05), as well as interleukin 6 secretion caused by thrombin (P < .005).Conclusions: This study demonstrates that endothelial cell activation by thrombin and downstream inflammatory responses can be inhibited by aprotinin in vitro through blockade of protease-activated receptor 1. Our results provide a new molecular 41 basis to help explain the anti-inflammatory properties of aprotinin reported clinically.