Molecular Mechanism of Thrombin Inhibition

凝血酶抑制的分子机制

• 批准号: 6671362
• 负责人: SCOTT T COOPER
• 金额: $ 12.08万
• 依托单位: UNIVERSITY OF WISCONSIN LA CROSSE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6671362
• 关键词: antifibrinolytic agents; antithrombins; blood coagulation; computer simulation; molecular dynamics; protease inhibitor; protein C; protein protein interaction; protein structure; protein structure function; serine proteinases; thrombin; thrombomodulin

DESCRIPTION (provided by applicant): The regulation of coagulation is central to many diseases, including heart disease and stroke. The objective of this research is to better understand the different mechanisms by which the serpins antithrombin (AT) and protein C inhibitor (PCI) inhibit free thrombin and thrombin bound to thrombomodulin (TM). Specifically the role of the H-helix, and other predicted contact points between the serpin and TM will be investigated. Previously we made the observation that, unlike AT, PCI is a potent inhibitor of thrombin bound to TM. The heparin binding domains of PCI and AT also differ; in AT the D-helix is a major part of the heparin-binding domain, while the H-helix is the heparin-binding domain in PCI. Alignment of the sequences of PCI, AT and heparin cofactor II (HCII) suggests that AT is unique in having a negatively charged H-helix, while the other serpins have positively charged helices. In recent work we demonstrated that changing the charge of the H-helix of AT makes it behave more like PCI in inhibiting thrombin bound to either heparin or thrombomodulin. The crystal structure of thrombin complexed with TM has recently been solved. This structure was used to generate a molecular model of AT complexed with thrombin and TM to explain kinetic data design new experiments. The first aim of this proposal is to continue to explore the roles of the D and H helices of AT in the inhibition of thrombin in the presence and absence of TM or heparin. The molecular model of a complex between thrombin, TM and AT revealed several other potential contact points between AT and TM, which would not be present between PCI and TM. In addition, the amino terminus of AT contains several more amino acids than does PCI, forming a loop which appears to sterically interfere with TM bound to thrombin. The second aim is to explore the importance of this loop by removing amino acid residues from the center of the loop and assaying the ability of these mutants to inhibit thrombin bound to TM. Another contact point in the complex is between AT residues R259 to R262 and three negatively charged amino acids on TM (E357, D398 and EH00). The third aim is to change these residues and measure the impact on inhibition of thrombin bound to TM The outcome of these experiments will provide a clearer understanding of the different mechanisms by which PCI and AT inhibit thrombin complexed with TM.

描述（由申请人提供）：凝血的调节是许多疾病的核心，包括心脏病和中风。本研究的目的是更好地了解丝氨酸蛋白酶抑制剂抗凝血酶（AT）和蛋白C抑制剂（PCI）抑制游离凝血酶和凝血酶与血栓调节蛋白（TM）结合的不同机制。特别是H-螺旋的作用，以及其他预测的丝氨酸蛋白酶抑制剂和TM之间的接触点将进行研究。以前我们观察到，与AT不同，PCI是与TM结合的凝血酶的有效抑制剂。PCI和AT的肝素结合结构域也不同;在AT中，D-螺旋是肝素结合结构域的主要部分，而H-螺旋是PCI中的肝素结合结构域。PCI，AT和肝素辅因子II（HCII）的序列的比对表明，AT是唯一的具有带负电荷的H-螺旋，而其他丝氨酸蛋白酶抑制剂具有带正电荷的螺旋。在最近的工作中，我们证明了改变AT的H-螺旋的电荷使其在抑制与肝素或血栓调节蛋白结合的凝血酶方面表现得更像PCI。凝血酶与TM复合物的晶体结构最近已得到解决。这种结构被用来产生一个分子模型的AT与凝血酶和TM复合来解释动力学数据设计新的实验。本提案的第一个目的是继续探索AT的D和H螺旋在存在和不存在TM或肝素的情况下抑制凝血酶的作用。凝血酶、TM和AT之间复合物的分子模型揭示了AT和TM之间的其他几个潜在接触点，这些接触点在PCI和TM之间不存在。此外，AT的氨基末端含有比PCI多几个氨基酸，形成一个环，该环似乎在空间上干扰TM与凝血酶的结合。第二个目标是通过从环中心去除氨基酸残基并测定这些突变体抑制与TM结合的凝血酶的能力来探索该环的重要性。复合物中的另一个接触点在AT残基R259至R262与TM上的三个带负电荷的氨基酸（E357、D398和EH 00）之间。第三个目的是改变这些残基并测量对抑制与TM结合的凝血酶的影响。这些实验的结果将提供PCI和AT抑制与TM复合的凝血酶的不同机制的更清楚的理解。

项目成果

Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition.

抗凝血酶中 H 螺旋的突变会降低肝素对蛋白酶抑制的刺激。

• DOI: 10.1016/j.bbapap.2007.08.020
• 发表时间: 2007
• 期刊: Biochimica et biophysica acta
• 作者: Gonzales,PatrickR;Walston,TimothyD;Camacho,LaureanoO;Kielar,DanaM;Church,FrankC;Rezaie,AlirezaR;Cooper,ScottT
• 通讯作者: Cooper,ScottT

Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin.

血栓调节蛋白通过提供丝氨酸蛋白酶抑制剂的结合位点和变构调节凝血酶的活性来增强凝血酶与蛋白 C 抑制剂的反应性。

• DOI: 10.1074/jbc.m307243200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Yang,Likui;Manithody,Chandrashekhara;Walston,TimothyD;Cooper,ScottT;Rezaie,AlirezaR
• 通讯作者: Rezaie,AlirezaR