Reuning U, Bang N U
Lilly Laboratories for Clinical Research, Eli Lilly & Company, Indianapolis, IN 46285.
Arterioscler Thromb. 1992 Oct;12(10):1161-70. doi: 10.1161/01.atv.12.10.1161.
The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.
在牛主动脉的培养平滑肌细胞（SMC）上证实了尿激酶型纤溶酶原激活物受体（u-PAR）。125I-尿激酶型纤溶酶原激活物（u-PA）的结合呈浓度依赖性，且在45 - 60分钟内可饱和。在功能性纤溶酶原激活研究中也发现了类似的浓度和时间依赖性。人双链高分子量u-PA及其酶原（pro-u-PA）以相同的亲和力（Kd）特异性结合。pro-u-PA与SMC结合后激活作用强烈加速，且仅在细胞表面存在纤溶酶原时发生。100倍摩尔过量的未标记高分子量u-PA可有效阻断放射性标记配体的结合；组织型纤溶酶原激活物、纤溶酶原、低分子量u-PA及无关蛋白则不能。针对负责u-PAR结合的u-PA特定序列的单克隆抗体可消除125I-u-PA的结合。SMC暴露于磷脂酰肌醇特异性磷脂酶C后，u-PA的结合急剧下降，证实了u-PAR的糖脂细胞锚定。牛和人α-凝血酶（240 nM）使125I-u-PA的结合增加了五倍，导致每个细胞的位点数量从约10^5增加到5×10^5，而Kd（1.29±0.39 nM）无显著变化。D-苯丙氨酸-脯氨酸-精氨酸-氯甲基酮对凝血酶活性位点的阻断导致刺激活性完全丧失，使用无活性的活性位点凝血酶突变体S205A时也是如此。水蛭素（100 μM）可阻断凝血酶的阴离子结合外位点，从而阻断u-PAR刺激活性。因此，催化活性和外位点的完整性对凝血酶的刺激活性均很重要。其他SMC有丝分裂原（表皮生长因子、转化生长因子-β1、碱性成纤维细胞生长因子、血小板衍生生长因子和佛波醇12-肉豆蔻酸酯13-乙酸酯）使SMC上的u-PAR表达增加6至20倍，同时使Kd增加4至10倍。在所有情况下，u-PAR的诱导均依赖于从头合成蛋白质。这些观察结果表明凝血酶和其他有丝分裂原在u-PAR调节中可能发挥作用，从而影响对SMC迁移和动脉粥样斑块形成很重要的细胞周围蛋白水解过程。
