REGULATION OF CELL SURFACE PLASMINOGEN ACTIVATION

通过结缔组织基质的细胞侵入需要蛋白酶 级联，其控制特征是调节激活 尿激酶型纤溶酶原激活剂（u-PA）。 U-PA是 合成为最低活性单链分子（scu-PA），但可以 被纤溶酶切割成活性双链形式（tcu-PA）。 这 循环，但基本过程一直令人困惑：首先，如果tcu-PA是 需要激活纤溶酶原，但纤溶酶需要激活scu- PA，这个过程是如何开始的？ 第二，因为这是一个“积极的 反馈回路“这个过程如何停止？此外，细胞上的tcu-PA 纤溶酶原激活剂可有效抑制u-PA受体（u-PAR 抑制剂（派）。那么细胞是如何通过富含PAI的环境侵入的呢？我们 已经使用了一种不被纤溶酶裂解的scu-PA形式，glu 158-scu-PA， 证明i）scu-PA表现出酶活性，并且当它 和纤溶酶原结合到它们各自的细胞表面结合位点， 纤溶酶的产生比反应物 在溶液中;这种活化是通过单链而不是两链进行 u-PA。 因此scu-PA可以引发该过程;和ii）scu-PA，在溶液中 可逆性结合派-2，而u-PA受体上的scu-PA不可逆性结合PAI-2。 压抑 因此纤溶酶原激活需要scu-PA首先 与派-2解离，然后与细胞表面受体结合，表达 甚至在过量派-2的存在下也具有酶活性。 这可能 影响起始过程，并允许细胞通过派侵入， 丰富的环境。 在这个应用程序中，我们将重点放在测试，每个 细胞表面纤溶酶原激活起始步骤的组分。 具体来说，我们建议确定。 1)是否可逆结合 PAI-2和/或u-PAR的scu-PA的表达在PAI-2和/或u-PAR的启动中具有调节作用。 细胞表面纤溶酶原激活（即，是scu-PA的分配 派2与u-PAR之间的关系是细胞表面纤溶酶原的主要决定因素 激活？）2)纤溶酶原被scu-PA激活的机制 100-在单核细胞表面比在溶液中更有效地折叠。 这是由于对K（m），K（cat）的影响，纤溶酶原作为一种 基质，或这些的组合？3)与纤溶酶原类似 激活纤维蛋白凝块是否发挥纤溶酶的酶活性 在加速细胞表面纤溶酶原活化中的作用。 这 工作应确定启动这一基本的管理机制， 入侵细胞的特性。

Cell invasion through connective tissue matrices requires a proteinase cascade, the controlling feature of which is regulated activation of plasminogen by urokinase type plasminogen activator (u-PA). U-PA is synthesized as a minimally active single chain molecule (scu-PA), but can be cleaved to an active two chain form (tcu-PA) by plasmin. This circular, but fundamental process has been puzzling: First, if tcu-PA is required to activate plasminogen, but plasmin is required to activate scu- PA, how does the process start? Second, because this is a "positive feedback loop." how does the process stop? Furthermore, tcu-PA on the cell u-PA receptor (u-PAR) is efficiently inhibited by plasminogen activator inhibitor (PAI). so how do cells invade through PAI-rich environments? We have used a form of scu-PA, glu158-scu-PA, not cleaved by plasmin to demonstrate that i) scu-PA exhibits enzymatic activity, and that when it and plasminogen are bound to their respective cell surface binding sites, plasmin is generated 100-fold more efficiently than if the reactants are in solution; this activation is carried out by single chain, not two chain u-PA. Hence scu-PA can initiate the process; and ii) scu-PA, in solution binds reversibly to PAI-2, whereas scu-PA on the u-PA receptor is not inhibited. Hence plasminogen activation requires that scu-PA first dissociate from PAI-2, then bind to cell surface receptor to express enzymatic activity even in the presence of excess PAI-2. This probably influences the initiation process, and allows cells to invade through PAI- rich environments. In this application we will focus on testing, each component of the initiating steps of cell surface plasminogen activation. Specifically, we propose to determine. 1) whether the reversible binding of scu-PA to PAI-2 and/or u-PAR has a regulatory role in the initiation of cell surface plasminogen activation (i.e.. is the partitioning of scu-PA between PAI 2 and u-PAR a primary determinant of cell surface plasminogen activation?) 2) The mechanism by which plasminogen is activated by scu-PA 100-fold more efficiently on the monocyte cell surface than in solution. Is it due to an effect on K(m), K(cat), the nature of plasminogen as a substrate, or a combination of these? 3) By analogy to plasminogen activation on fibrin clots whether the enzymatic activity of plasmin plays a role in accelerating plasminogen activation at the cell surface. This work should define regulatory mechanisms for initiating this fundamental property of invading cells.