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

血液凝块的形成是对损伤的一种非常重要的正常生理反应。如果没有足够的凝血，损伤可能会导致有害或致命的出血。然而，硬币的另一面是，太多的凝血，或太容易引发的凝血，会导致血液凝块的形成，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。这种导致疾病的凝血被称为血栓形成，它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，血液凝结的过程必须得到精细的调控，以允许凝血来止血，同时避免致命的血液凝块的形成。在血液凝结过程中，有两种关键机制在起作用。一种是被称为血小板的非常小的血细胞的激活，它们聚集(聚集在一起)并在凝血过程中提供第一反应。第二种是凝血酶将一种名为纤维蛋白原的血液蛋白质转化为纤维蛋白。纤维蛋白自发聚合成由纤维组成的网络。纤维蛋白纤维网络与激活的血小板相互作用，形成血栓。已知纤维蛋白通过血小板上一种称为GPIIb/IIIa整合素的受体与血小板相互作用。然而，新的研究表明，还有第二种受体与血小板上的纤维蛋白结合。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，以及这种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一个特殊部分与纤维蛋白结合，称为αC区。此外，我们已经开始开发和鉴定新的构象蛋白，称为亲和蛋白，能够与GPVI结合，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(亲和分子)来阻断这种相互作用，以全面描述GPVI-纤维蛋白相互作用在血栓形成中的作用。在我们研究项目的第一部分，我们将筛选大量与GPVI结合的亲和分子。接下来，我们将筛选正结合仿射体是否能阻断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(聚集)、血栓形成和血栓生长的体外模型来测试封闭仿射器。在第三部分中，我们将使用质谱学、定点突变和X射线结晶学的方法，在非常详细的分子水平上表征仿射分子与GPVI的相互作用。这将有助于我们了解亲和分子如何与GPVI相互作用，以及纤维蛋白如何间接与GPVI相互作用。总之，这些研究将加强我们对血栓形成这一重要的新途径的了解，并可能有助于找到新的方法来预防有心脏病发作、中风、深静脉血栓形成或肺血栓形成风险的患者的致命血栓。

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.