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Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis
发布于：2013年4月8日 文字：【大】【中】【小】
PLoS One. 2013;8(3):e59372. doi: 10.1371/journal.pone.0059372. Epub 2013 Mar 29.
Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.
Source
Department of Bioengineering, RiceUniversity,Houston,Texas,United States of America.
Abstract
BACKGROUND:
Vascular endothelial cells (ECs) express and release protein components of the complement pathways, as well as secreting and anchoring ultra-large von Willebrand factor (ULVWF) multimers in long string-like structures that initiate platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP) is an important non-antibody-requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the AP (atypical hemolytic-uremic syndrome) or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted by/anchored to ECs (thrombotic thrombocytopenic purpura). Our goal was to determine if EC-anchored ULVWF strings caused the assembly and activation of AP components, thereby linking two essential defense mechanisms.
METHODOLOGYPRINCIPAL FINDINGS:
We quantified gene expression of these complement components in cultured human umbilical vein endothelial cells (HUVECs) by real-time PCR: C3 and C5; complement factor (CF) B, CFD, CFP, CFH and CFI of the AP; and C4 of the classical and lectin (but not alternative) complement pathways.
We used fluorescent microscopy, monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of >150 images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored to, the HUVECs (under conditions of ADAMTS-13 inhibition). We found that HUVEC-released C4 did not attach to ULVWF strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes. This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb) on ULVWF strings.
CONCLUSIONSSIGNIFICANCE:
AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric strings. Our findings provide one possible molecular mechanism for clinical linkage between different types of thrombotic and complement-mediated disorders.
锚定在内皮细胞表面的超大vWF分子上的补体旁路蛋白的聚集和激活是补体系统和凝血系统的连接点
背景：血管内皮细胞(ECs)可以表达和释放补体蛋白，同时也可以分泌和锚定超大vWF分子(ULvWF)多聚物。ULvWF为长线样结构，可以在止血和血栓形成过程中引发血小板的聚集。补体旁路途径是一种重要的非抗体依赖的防御系统。血栓性微血管病发病机制与补体旁路途径失调(非典型性溶血尿毒综合征)或ADAMTS-13 对血管内皮细胞分泌或者锚定的线样ULvWF剪切不足(血栓性血小板减少性紫癜)有关。本研究目的在于阐明血管内皮细胞锚定的ULvWF是否可以导致补体旁路途径蛋白的聚集和激活，进而寻找两个重要的防御系统的联系。
方法和结果：本研究应用实时定量PCR法检测了体外培养的人脐静脉内皮细胞补体蛋白的基因表情况，包括旁路途径蛋白C3，C5，B因子，D因子，P因子，H因子和I因子，以及经典和凝集素途径蛋白C4。本研究采用荧光显微镜，补体蛋白特异性抗体和荧光标记的二抗，定量分析至少150张图像，从而研究人脐静脉内皮细胞表达的补体蛋白与内皮细胞表达或者锚定的ULvWF的关系(以上实验均在ADAMTS-13活性被抑制的条件下进行)。我们发现，人脐静脉内皮细胞释放的C4不与线样ULvWF粘附。但C3， B因子，D因子，P因子，C5，H因子和I因子均可与线样ULvWF粘附，且粘附量与补体激活程度相关。以上结果被进一步证实：失活的B因子可以阻断线样ULvWF上旁路途径C3转化酶(C3bBb)的形成。
结论：旁路途径蛋白可以在内皮细胞表达或锚定的线样ULvWF多聚物上聚集和激活。本研究结果提示了两种不同的补体介导的血栓性疾病临床关联的一种可能的分子机制。