STRUCTURAL STUDIES ON SERINE PROTEASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该提案的重点是生物学相关的丝氨酸蛋白酶与抑制剂，底物和效应物的复合物，我们目前缺乏结构信息。重点主要是凝血酶和活化蛋白C，这两种关键蛋白酶参与血液凝固的进展（凝血酶）和抑制（活化蛋白C）。 我们计划解决一些尚未解决的问题，这些问题将从晶体结构的可用性中受益匪浅。在凝血酶的情况下，我们计划结晶的酶与血栓调节蛋白和蛋白C的复合物，以建立其抗凝活性的分子基础。我们计划结晶凝血酶突变体W215 A/E217 A，这是进入I期临床试验，在复杂的血小板受体GPIb，以确定这种新发现的相互作用的结合模式。我们计划结晶凝血酶原，以获得信息的结构的酶原形式的凝血酶。我们还计划结晶与GPIb和蛋白酶激活受体PAR 1、PAR 3和PAR 4、血栓调节蛋白和蛋白C的片段复合的、沿着凝血酶原途径沿着最相关的中间体美佐凝血酶。在活化蛋白C的情况下，我们计划将酶结晶为E*、E和E：Na+形式，并与PAR 1和凝血因子Va的片段复合。这些蛋白质及其复合物的结构将产生重大进展，在我们的理解的分子基础上的凝血酶促凝血，促血栓形成和抗凝血的活动，以及酶的方式激活凝血酶原通过meizothrobin。结合GPIb的W215 A/E217 A的结构将揭示这种临床相关突变体显著抗血栓形成作用的分子基础。活化蛋白C的结构将促进我们对这种酶在控制血栓形成和炎症中的功能的理解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal focuses on biologically relevant serine proteases in complex with inhibitors, substrates and effectors for which we currently lack structural information. The focus is mainly on thrombin and activated protein C, two key proteases involved in the progression (thrombin) and inhibition (activated protein C) of blood coagulation. We plan to address a number of unsolved issues that will benefit tremendously from the availability of crystal structures. In the case of thrombin, we plan to crystallize the enzyme in complex with thrombomodulin and protein C to establish the molecular basis of its anticoagulant activity. We plan to crystallize the thrombin mutant W215A/E217A, that is entering Phase I clinical trials, in complex with the platelet receptor GPIb to identify the mode of binding underscoring this newly discovered interaction. We plan to crystallize prothrombin in order to obtain information on the architecture of the zymogen form of thrombin. We also plan to crystallize meizothrombin, the most relevant intermediate along the prothrombin pathway, in complex with GPIb and fragments of the protease activated receptors PAR1, PAR3 and PAR4, thrombomodulin and protein C. In the case of activated protein C, we plan to crystallize the enzyme in the E*, E and E:Na+ forms and in complex with fragments of PAR1 and coagulation factor Va. Structures of these proteins and their complexes will produce major advances in our understanding of the moelcular basis of thrombin procoagulant, prothrombotic and anticoagulant activities in the blood, and the way the enzyme is activated from prothrombin via meizothrombin. The structure of W215A/E217A bound to GPIb will reveal the molecular basis of the remarkable antithrombotic effect of this clinically relevant mutant. Structures of activated protein C will advance our understanding of the function of this enzyme in the control of thrombosis and inflammation.