Zucker S, Conner C, DiMassmo B I, Ende H, Drews M, Seiki M, Bahou W F
Department of Medicine and Research, Department of Veterans Affairs Medical Center, Northport, New York 11768, USA.
J Biol Chem. 1995 Oct 6;270(40):23730-8. doi: 10.1074/jbc.270.40.23730.
Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of alpha-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metaloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.
血管生成需要在内皮细胞迁移和增殖之前降解血管基底膜；蛋白酶是这一过程中的关键成分。由于凝血酶对内皮细胞有多种作用，我们利用人脐静脉内皮细胞研究了其在基质金属蛋白酶激活中的作用。内皮细胞条件培养基的明胶酶谱显示有一条明显的72 kDa前明胶酶A条带。向内皮细胞中添加α-凝血酶会产生64 kDa和62 kDa的明胶水解条带，这与前明胶酶A的激活一致；在无细胞的情况下凝血酶没有作用。这种作用需要凝血酶的蛋白水解位点，因为凝血酶的特异性抑制剂可消除前明胶酶A的激活。基质金属蛋白酶抑制剂可减少凝血酶诱导的前明胶酶A的激活。用过量的金属蛋白酶组织抑制剂-2或前明胶酶A的COOH末端片段预处理内皮细胞可消除凝血酶介导的前明胶酶A的激活，推测这是通过与天然前明胶酶A的COOH末端竞争与内皮细胞质膜上的激活位点相互作用来实现的。尽管通过Northern和Western印迹法在内皮细胞中证实了膜型基质金属蛋白酶的存在，但该分子在凝血酶诱导的前明胶酶A激活中的受体功能仍需阐明。前明胶酶A的激活不需要由凝血酶受体介导的细胞内信号转导事件。这些数据表明：1）内皮细胞表达一种新的前明胶酶A激活机制；2）具有蛋白水解活性的凝血酶调节这种激活机制；3）激活独立于功能性凝血酶受体发生。
