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Conformational proteins to study molecular mechanisms underpinning GPVI-fibrin(ogen) interaction in blood clot propagation

构象蛋白研究血凝块传播中 GPVI-纤维蛋白（原）相互作用的分子机制

基本信息

批准号：
BB/W000237/1
负责人：
Robert Ariens
金额：
$ 85.2万
依托单位：
University of Leeds
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW000237%2F1
关键词：
Conformational proteins study molecular mechanisms

项目摘要

The formation of a blood clot is a very important normal physiological response to injury. Without adequate clotting, injury could lead to harmful or deadly bleeding. Yet, the flip side of the coin is that too much clotting, or clotting that is too easily triggered, can lead to formation of blood clots that block important blood vessels in the heart (heart attack), brain (stroke), legs (deep vein thrombosis) or lungs (pulmonary embolism). This disease-causing clotting is called thrombosis, which is one of the leading causes of mortality in the developed and developing world. Understandably the process of blood clotting has to be finely regulated to allow for clotting to stem bleeding yet avoid deadly blood clots from developing. Two key mechanisms are at play during blood clotting. One is the activation of very small blood cells called platelets which aggregate (clump together) and provide the first response during clotting. The second is the conversion of a blood protein called fibrinogen to fibrin by thrombin. Fibrin spontaneously polymerises into a network made of fibres. The fibrin fibre network interacts with the activated platelets to form the blood clot. It was known that fibrin interacts with platelets through a receptor called GPIIb/IIIa integrin on the platelets. However, new research has shown that there is a second receptor that binds fibrin on the platelet. This receptor is called GPVI and is a member of the immunoglobulin superfamily. The mechanisms by which these two proteins (fibrin and GPVI) interact, and how the interaction contributes to thrombosis, are poorly understood. We have recently found that GPVI binds to fibrin via a particular part of the fibrin molecule, called the alphaC-region. In addition, we have started developing and characterising new conformational proteins called Affimers that are able to bind GPVI and displace fibrin from GPVI and block it from binding. Our BBSRC research proposal aims to fully characterise the role of GPVI-fibrin interaction in thrombosis using conformational proteins (Affimers) to block this interaction. In the first part of our research project, we will screen a very large number of Affimers for binding to GPVI. Next, we will screen whether the positive binding Affimers block the interaction between GPVI and fibrin. In the second part, we will test blocking Affimers using in-vitro models of platelet aggregation (clumping), thrombus formation and thrombus growth. In the third part, we will characterise the interaction of the Affimers and GPVI at a very detailed molecular level using mass spectrometry, site-directed mutagenesis and X-ray crystallography methods. This will help us to understand how the Affimer interacts with GPVI, and indirectly how fibrin interacts with GPVI. Together, these studies will enhance our understanding of this important novel pathway of thrombus formation and may help to find new ways of preventing deadly blood clots in patients at risk of a heart attack, stroke, deep vein thrombosis or pulmonary emblism.

血凝块的形成是对损伤的非常重要的正常生理反应。如果没有足够的凝血，损伤可能导致有害或致命的出血。然而，硬币的另一面是，太多的凝血，或太容易触发的凝血，可能导致血栓形成，阻塞心脏（心脏病发作），大脑（中风），腿部（深静脉血栓形成）或肺部（肺栓塞）的重要血管。这种引起疾病的凝血被称为血栓形成，这是发达国家和发展中国家死亡的主要原因之一。可以理解的是，血液凝固的过程必须被精细地调节，以允许凝血来阻止出血，同时避免致命的血栓形成。在血液凝固过程中有两种关键机制起作用。一种是激活非常小的血细胞，称为血小板，血小板聚集在一起，并在凝血过程中提供第一个反应。第二种是凝血酶将一种叫做纤维蛋白原的血液蛋白转化为纤维蛋白。纤维素自发地聚合成纤维网络。纤维蛋白纤维网络与活化的血小板相互作用以形成血凝块。已知纤维蛋白通过血小板上称为GPIIb/IIIa整联蛋白的受体与血小板相互作用。然而，新的研究表明，有第二种受体结合血小板上的纤维蛋白。这种受体被称为GPVI，是免疫球蛋白超家族的成员。这两种蛋白质（纤维蛋白和GPVI）相互作用的机制，以及相互作用如何导致血栓形成，目前还知之甚少。我们最近发现GPVI通过纤维蛋白分子的特定部分（称为α C区）与纤维蛋白结合。此外，我们已经开始开发和表征新的构象蛋白质，称为Affimers，能够结合GPVI并取代GPVI中的纤维蛋白并阻止其结合。我们的BBSRC研究计划旨在使用构象蛋白（Affimers）来阻断GPVI-纤维蛋白相互作用在血栓形成中的作用。在我们的研究项目的第一部分，我们将筛选大量的Affimers与GPVI结合。接下来，我们将筛选阳性结合Affimers是否阻断GPVI和纤维蛋白之间的相互作用。在第二部分中，我们将使用血小板聚集（结块）、血栓形成和血栓生长的体外模型来测试阻断Affimers。在第三部分中，我们将使用质谱，定点突变和X射线晶体学方法在非常详细的分子水平上研究Affimers和GPVI的相互作用。这将有助于我们了解Affimer如何与GPVI相互作用，以及纤维蛋白如何间接与GPVI相互作用。总之，这些研究将增强我们对血栓形成这一重要的新途径的理解，并可能有助于找到预防心脏病发作，中风，深静脉血栓形成或肺栓塞风险患者致命血栓的新方法。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Uncertainties about the roles of anticoagulation and microclots in postacute sequelae of severe acute respiratory syndrome coronavirus 2 infection.

抗凝和微凝块在严重急性呼吸综合征冠状病毒 2 感染急性后遗症中作用的不确定性。

DOI：
10.1016/j.jtha.2023.07.012
发表时间：
2023
期刊：
JTH
影响因子：
0
作者：
Connors JM
通讯作者：
Connors JM

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

DOI：
10.1371/journal.ppat.1011529
发表时间：
2023-07
期刊：
PLOS PATHOGENS
影响因子：
6.7
作者：
Pierce, Danielle M.;Buchanan, Frazer J. T.;Macrae, Fraser L.;Mills, Jake T.;Cox, Abigail;Abualsaoud, Khadijah M.;Ward, Joseph C.;Ariens, Robert A. S.;Harris, Mark;Stonehouse, Nicola J.;Herod, Morgan R.
通讯作者：
Herod, Morgan R.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure.

纤维蛋白-糖蛋白 VI 相互作用增加血小板促凝血活性并影响凝块结构。