Hajjar K A, Harpel P C, Jaffe E A, Nachman R L
J Biol Chem. 1986 Sep 5;261(25):11656-62.
Endothelial cells are known to release the two major forms of plasminogen activator, tissue plasminogen activator (TPA) and urokinase. We have previously demonstrated that plasminogen (PLG) immobilized on various surfaces forms a substrate for efficient conversion to plasmin by TPA (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, P. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human PLG to cultured human umbilical vein endothelial cell (HUVEC) monolayers, utilizing a newly devised cell monolayer enzyme-linked immunosorbent assay system. PLG binding to HUVEC was concentration dependent and saturable at physiologic PLG concentration (2 microM). Binding of PLG was 70-80% inhibited by 10 mM epsilon-aminocaproic acid, suggesting that it is largely mediated by the lysine-binding sites of PLG. PLG bound at an intermediate level to human fibroblasts, poorly to human smooth muscle cells, and not at all to bovine smooth muscle or bovine endothelial cells; unrelated proteins such as human albumin and IgG failed to bind HUVEC. PLG binding to HUVEC was rapid, reaching a steady state within 20 min, and quickly reversible. 125I-PLG bound to HUVEC with an estimated Kd of 310 +/- 235 nM (S.E.); each cell contained 1,400,000 +/- 1,000,000 (S.E.) binding sites. Functional studies demonstrated that HUVEC-bound PLG is activatable by TPA according to Michaelis-Menten kinetics (Km, 5.9 nM). Importantly, surface-bound PLG was activated with a 12.7-fold greater catalytic efficiency than fluid phase PLG. These results indicate that PLG binds to HUVEC in a specific and functional manner. Binding of PLG to endothelial cells may play a pivotal role in modulating thrombotic events at the vessel surface.
已知内皮细胞可释放两种主要形式的纤溶酶原激活剂，即组织纤溶酶原激活剂（TPA）和尿激酶。我们之前已经证明，固定在各种表面的纤溶酶原（PLG）可形成一种底物，通过TPA高效转化为纤溶酶（Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, P. C. (1985) J. Biol. Chem. 260, 10346 - 10352）。我们现在报告利用新设计的细胞单层酶联免疫吸附测定系统，人PLG与培养的人脐静脉内皮细胞（HUVEC）单层的结合情况。PLG与HUVEC的结合呈浓度依赖性，在生理PLG浓度（2 microM）时可饱和。10 mM ε-氨基己酸可抑制70 - 80%的PLG结合，这表明其主要由PLG的赖氨酸结合位点介导。PLG与人类成纤维细胞呈中等水平结合，与人类平滑肌细胞结合较差，与牛平滑肌或牛内皮细胞完全不结合；诸如人白蛋白和IgG等无关蛋白质不能结合HUVEC。PLG与HUVEC的结合迅速，在20分钟内达到稳态，且快速可逆。125I - PLG与HUVEC结合，估计解离常数（Kd）为310 +/- 235 nM（标准误）；每个细胞含有1,400,000 +/- 1,000,000（标准误）个结合位点。功能研究表明，根据米氏动力学（Km，5.9 nM），HUVEC结合的PLG可被TPA激活。重要的是，表面结合的PLG的催化效率比液相PLG高12.7倍。这些结果表明PLG以一种特异性和功能性的方式与HUVEC结合。PLG与内皮细胞的结合可能在调节血管表面的血栓形成事件中起关键作用。
