Ugwu F, Lemmens G, Collen D, Lijnen H R
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Thromb Haemost. 1999 Sep;82(3):1127-31.
Stromelysin-1 (MMP-3) cleaves a 55 kDa kringle 1-4 fragment, containing the lysine-binding site(s) involved in cellular binding, from 92 kDa plasminogen and removes a 17 kDa NH2-terminal fragment, containing the cellular receptor-binding site, from 45 kDa urokinase (u-PA), but a potential role of MMP-3 in the regulation of cellular fibrinolytic activity by affecting binding and/or activation of plasminogen and/or single-chain u-PA has not been established. Human plasminogen (input concentration 100 nM for 4x10(6) cells per ml) was shown to bind specifically to human monocytoid THP-1 cells, to murine MMP-3 deficient smooth muscle cells (SMC) and fibroblasts (1.9, 0.92 and 1.0x10(6) molecules per cell, respectively). Treatment with MMP-3 (final concentration 0-50 nM) of cells saturated with bound plasminogen (about 25 nM), overnight at 37 degrees C, resulted in a dose-dependent reduction of the amount of u-PA activatable plasminogen (reduction to 25-40% of the value in the absence of MMP-3). Immunoblotting with specific monoclonal antibodies and autoradiography of eluates of the cells treated with MMP-3 revealed cleavage of plasminogen into the 55 kDa fragment and miniplasminogen (kringle 5 plus the proteinase domain). Binding of human single chain u-PA (scu-PA) to human THP-1 and HT 1080 cells amounted to 2.5x10(6) and 7.1x10(6) molecules per cell, respectively. Treatment with MMP-3 (final concentration 0-25 nM) of cell-bound u-PA (about 17 nM for THP-1 and 47 nM for HT1080 cells), overnight at 37 degrees C, did not alter cell-associated u-PA activity, measured in a direct chromogenic substrate assay or in a plasminogen-coupled chromogenic substrate assay (residual u-PA activity always > or =85% of that without MMP-3 treatment). Autoradiography of 125I-labeled u-PA moieties, removed from the cells by treatment with acid or with phosphatidylinositol phospholipase C, confirmed that u-PA remained essentially intact after MMP-3 treatment. These data indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, without affecting cell-bound u-PA activity.
基质溶素-1（MMP-3）从92 kDa的纤溶酶原上切割下一个55 kDa的kringle 1-4片段，该片段包含参与细胞结合的赖氨酸结合位点，并且从45 kDa的尿激酶（u-PA）上切除一个17 kDa的NH2末端片段，该片段包含细胞受体结合位点，但是MMP-3在通过影响纤溶酶原和/或单链u-PA的结合和/或激活来调节细胞纤溶活性方面的潜在作用尚未明确。已证明人纤溶酶原（每毫升4×10(6)个细胞的输入浓度为100 nM）能特异性结合人单核细胞样THP-1细胞、小鼠MMP-3缺陷型平滑肌细胞（SMC）和成纤维细胞（分别为每个细胞1.9、0.92和1.0×10(6)个分子）。用MMP-3（终浓度0 - 50 nM）处理已饱和结合纤溶酶原（约25 nM）的细胞，在37℃下过夜，导致可被u-PA激活的纤溶酶原量呈剂量依赖性减少（减少至无MMP-3时值的25 - 40%）。用特异性单克隆抗体进行免疫印迹以及对用MMP-3处理的细胞洗脱液进行放射自显影，显示纤溶酶原被切割成55 kDa片段和微型纤溶酶原（kringle 5加上蛋白酶结构域）。人单链u-PA（scu-PA）与人THP-1细胞和HT 1080细胞的结合量分别为每个细胞2.5×10(6)和7.1×10(6)个分子。用MMP-3（终浓度0 - 25 nM）处理细胞结合的u-PA（THP-1细胞约为17 nM，HT1080细胞约为47 nM），在37℃下过夜，在直接显色底物测定或纤溶酶原偶联显色底物测定中，未改变细胞相关的u-PA活性（残余u-PA活性始终≥无MMP-3处理时的85%）。用酸或磷脂酰肌醇磷脂酶C处理从细胞上移除的125I标记的u-PA部分的放射自显影证实，MMP-3处理后u-PA基本保持完整。这些数据表明，MMP-3可能通过减少可激活的纤溶酶原量来下调细胞相关的纤溶活性，而不影响细胞结合的u-PA活性。
