REGULATION OF CELL SURFACE PLASMINOGEN ACTIVATION

细胞通过结缔组织基质的侵袭需要一种蛋白酶 级联，其控制特征是调节激活 纤溶酶原由尿激酶型纤溶酶原激活物(u-PA)激活。U-PA是 作为最低活性的单链分子(SCU-PA)合成，但可以 被纤溶酶切割成活性双链形式(TCuPA)。这 循环，但基本过程一直令人费解：首先，如果TCU-PA是 需要激活纤溶酶原，但需要纤溶酶激活SCU- 爸，这个过程是怎么开始的？第二，因为这是一个“积极的” 反馈环路。这一过程如何停止？此外，细胞上的TCU-PA 纤溶酶原激活剂对u-PA受体的有效抑制作用 抑制剂(PAI)。那么细胞是如何通过富含PAI的环境入侵的呢？我们 使用了一种形式的SCU-PA，Glu158-SCU-PA，未被纤溶酶切割 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部位， 产生纤溶酶的效率是反应物的100倍 在溶液中，这种激活是由单链进行的，而不是两条链 U-PA。因此，SCU-PA可以启动该过程；以及ii)SCU-PA，在溶液中 与PAI-2可逆结合，而u-PA受体上的SCU-PA不可逆结合 被禁止了。因此，纤溶酶原激活需要SCU-PA首先 解离PAI-2，然后与细胞表面受体结合表达 即使在存在过量PAI-2的情况下，酶活性也是如此。这很可能是 影响启动过程，并允许细胞通过PAI- 丰富的环境。在本应用程序中，我们将重点介绍测试，每个 细胞表面纤溶酶原激活的起始步骤的组成部分。 具体地说，我们建议确定。1)可逆绑定是否 SCU-PA到PAI-2和/或u-PAR在启动 细胞表面纤溶酶原激活(即是SCU-PA的分区 PAI-2与细胞表面纤溶酶原主要决定因素u-PAR之间的关系 激活？)2)SCU-PA激活纤溶酶原的机制 在单核细胞表面的效率是在溶液中的100倍。 这是由于对K(M)、K(CAT)的影响，纤溶酶原作为一种 底物，还是它们的组合？3)类似于纤溶酶原 纤溶酶活性对纤维蛋白凝块的激活作用 促进细胞表面纤溶酶原激活的作用。这 工作应该定义启动这一基本原则的监管机制 入侵细胞的属性。

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.