Yakovlev S, Mikhailenko I, Tsurupa G, Belkin A M, Medved L
Leonid Medved, PhD, University of Maryland School of Medicine, Center for Vascular and Inflammatory Diseases, 800 West Baltimore Street, Baltimore, MD, 21201, USA, Tel.: +1 410 706 8065, Fax: +1 410 706 8121, E-mail:
Thromb Haemost. 2014 Dec;112(6):1244-51. doi: 10.1160/TH14-01-0079. Epub 2014 Aug 21.
Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.
在纤维蛋白原转化为纤维蛋白的过程中，含有RGD识别基序的纤维蛋白原αC结构域形成有序的αC聚合物。我们之前的研究表明，这些结构域的聚合促进了内皮细胞的整合素依赖性黏附与铺展，以及整合素介导的FAK和ERK1/2信号通路的激活。本研究的主要目的是测试αC结构域聚合对伤口愈合过程中内皮细胞迁移和增殖的影响，并阐明αC聚合物对内皮细胞具有卓越活性的潜在机制。在体外伤口愈合试验中，用αC单体或αC聚合物包被的组织培养板上的汇合内皮细胞单层通过划痕造成损伤，并通过延时视频显微镜监测伤口闭合情况。尽管通过ELISA证实板上包被了等量的αC种类，但细胞在αC聚合物上的伤口闭合速度要快得多，这表明αC结构域的聚合促进了细胞迁移和增殖。同样，细胞增殖试验表明，内皮细胞在αC聚合物上的增殖也更有效。整合素阻断肽GRGDSP和ERK1/2信号通路的特异性拮抗剂对两种类型底物上的伤口闭合均有同等程度的抑制作用。相反，用特异性拮抗剂阻断FAK信号通路仅降低了αC聚合物上的伤口闭合。这些结果表明，αC结构域的聚合主要通过FAK信号通路增强整合素依赖性内皮细胞迁移和增殖。此外，αC聚合物中整合素结合RGD基序的聚集是触发这些事件的主要机制。
