Structure, function, and regulation of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)

凝血酶激活的纤维蛋白溶解抑制剂（TAFI）是在血浆中发现的调节血凝块分解（称为纤维蛋白溶解的过程）的蛋白质。我们的建议旨在研究TAFI如何被激活以及其活性如何被关闭的机制。这些事件共同控制TAFI在正常生理学（如预防出血）和疾病（如心脏病发作和中风）中的功能。**TAFI是一种酶的前体，需要被另一种酶切割以形成活化的TAFI（TAFIa）。能够激活TAFI的酶是凝血酶或纤溶酶，尽管这两种酶在这一作用中都非常低效。然而，当凝血酶与称为血栓调节蛋白（TM）的细胞表面辅因子相互作用时，它激活TAFI的效率增加了一千倍以上。虽然先前的研究已经定义了加速TAFI激活所必需的TM的特定部分，但尚不清楚这些部分如何有助于观察到的显着程度的加速。我们认为TM的某些部分与凝血酶相互作用，其他部分与TAFI相互作用。我们计划专注于TM的每一个部分。我们将在TM和TAFI中进行突变，以确定参与TAFI-TM相互作用的相应蛋白质中的确切氨基酸残基。我们将评估突变对TAFI激活速率和TM与TAFI结合能力的影响。此外，我们将突变TM中的一个关键残基，该残基影响TM如何与凝血酶相互作用，并且似乎可以区分凝血酶-TM的两种底物：TAFI和蛋白C。这些结构-功能研究将与分子建模分析相结合，以证实我们认为重要的各种氨基酸残基的作用。** 血液中形成的大多数酶都有匹配的抑制剂，可以快速而紧密地结合，以控制剩余的酶量。TAFIa是不寻常的，因为它缺乏这样的抑制剂。相反，TAFIa活性迅速衰减，在体温下半衰期仅为8 - 15分钟。先前的研究结果表明，TAFIa在失去活性时会发生很大的结构变化。已经确定了可能参与这种结构变化的蛋白质的一些区域，但它们的确切作用仍然是一个谜。此外，还有其他地区可能也有贡献，结构变化的细节仍有待确定。我们将鉴定TAFIa中通过突变特定氨基酸或氨基酸组来控制其失活速率的所有氨基酸。我们将使用一种称为氢-氘交换的技术（结构质谱法的一种形式），以便在真实的时间内跟踪TAFIa失去活性时的结构变化。 这些高度创新的研究将建立新的范式，为酶的不稳定性的结构基础的理解。

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a protein found in blood plasma that regulates blood clot breakdown, a process known as fibrinolysis. Our proposal seeks to investigate the mechanisms of how TAFI is activated and how its activity is turned off. Together, these events control the function of TAFI in normal physiology (such as prevention of bleeding) and in disease (such as the development of heart attack and strokes). ******TAFI is the precursor of an enzyme, and requires cleavage by another enzyme to form activated TAFI (TAFIa). The enzymes that are capable of activating TAFI are thrombin or plasmin, although both of these enzymes are very inefficient in this role. However, when thrombin interacts with a cell-surface cofactor called thrombomodulin (TM), the efficiency with which it activates TAFI increases by more than a thousand fold. While previous studies have defined particular parts of TM that are necessary to accelerate TAFI activation, it is not known how these parts contribute to the remarkable degree of acceleration that is observed. We believe that some parts of TM interact with thrombin, and other parts interact with TAFI. We plan to focus on each of these parts of TM. We will make mutations in both TM and in TAFI to define the exact amino acid residues in the respective proteins participate in the TAFI-TM interaction. We will assess the effect of the mutations on the rate of TAFI activation and on the ability of TM to bind to TAFI. In addition, we will mutate a key residue in TM that influences how TM interacts with thrombin and that appears to discriminate between the two substrates of thrombin-TM: TAFI and Protein C. These structure-function studies will be allied with molecular modeling analyses in order to corroborate the roles of the various amino acid residues we believe to be important. ******Most enzymes formed in the blood have matching inhibitors that bind rapidly and tightly to control the amount of enzyme that remains. TAFIa is unusual in that it lacks such an inhibitor. Instead, TAFIa activity rapidly decays with a half-life of only 8 15 minutes at body temperature. Previous findings have shown that TAFIa undergoes a large structural change as it loses activity. Some regions of the protein that may participate in this structural change have been identified, but their exact role remains a mystery. In addition, there are other regions that likely contribute, and the details of the structural change remain to be determined. We will identify all of the amino acids in TAFIa that control the rate of its inactivation by mutating particular amino acids or groups of amino acids. We will use a technique known as hydrogen-deuterium exchange (a form of structural mass spectrometry) in order to track, in real time, structural changes in TAFIa as it loses activity. These highly innovative investigations will establish new paradigms for the understanding of the structural basis for enzyme instability.