Inhibitors of Human Factor XIIIa as New Anticoagulants

摘要 我们研究的长期目标是开发不会导致出血的有效抗凝剂。 适用于更广泛的静脉血栓栓塞症(VTE)患者的安全使用。 该项目旨在通过靶向人类凝血因子XIIIA(FXIIIA)来开发有效和更安全的抗凝血剂。全 可用的抗凝剂与出血的重大风险有关。目前的抗凝剂直接抑制 或间接凝血酶和/或凝血因子Xa。这就是它们在临床上有效的原因，但这也是 它们导致出血的原因。中心假设是抑制FXIIIa将导致有效的 在不引起大量出血的情况下防止静脉血栓形成。与所有其他凝血因子形成对比的是 丝氨酸蛋白酶，FXIIIa是一种谷氨酰胺转氨酶，催化凝血过程的最后一步。这 FXIIIa的独特生化方面一直在VTE的背景下进行调查。体外实验 研究表明，用一种实验性的转谷氨酰胺酶抑制剂治疗正常人血液会增加红细胞 挤压收缩血块，缩小血块大小。各种研究还表明，某些FXIIIa 基因多态性对VTE有显著的保护作用，而杂合子FXIII缺陷小鼠则没有 有出血过多的迹象。因此，FXIIIa可能成为开发新的治疗靶点的潜在靶点。 对不会显著增加出血风险的VTE进行有效的治疗。尽管有这样的承诺，但很少有人 FXIIIa抑制剂已经被开发出来，所有这些都缺乏实质性的选择性，因为它们也可以抑制其他 转谷氨酰胺酶通过阻断其活性部位。因此，我提出了磺化非糖糖- 氨基多糖模拟作为开发FXIIIa抑制剂的平台。磺化分子是为了抑制FXIIIa 通过变构调节有效地和选择性地。在初步研究中，我发现了两种磺化 以低微摩尔势能抑制FXIIIa的分子。这两个分子抑制FXIIIa介导的 纤维蛋白聚合。这两个分子不影响其他凝血因子，也不影响血管活性。 三个细胞系。分子模型预测了这些分子在FXIIIa上可能的结合部位。在这 提议，我的具体目标是使用多学科方法来建立有效和 磺化分子对FXIIIa的选择性抑制我将综合两个“领先”的先进图书馆 并评估其作为抗凝剂的生物化学和生物学潜力。该提案是 创新是因为i)它提出了一种新的方法来克服目前VTE治疗的局限性；ii) 它利用多学科方法来调查具体目标；以及iii)它引入了新技术 具有专有的结构和机械方面。该项目还具有重要意义，因为它将：i)确定 2-3种有效的、特异的和变构的FXIIIa抑制剂，用于未来在VTE和出血动物模型中的评估； 二)提供新的工具，以更好地了解FXIIIa在凝血生理和病理中的作用；三)调查 通过变构调节FXIIIa的另一种方法，为转化抗凝剂铺平道路。

SUMMARY The long-term goal of our research is to develop effective anticoagulants that do not cause bleeding complications to be safely used for a wider range of patients suffering from venous thromboembolism (VTE). This project aims at developing effective and safer anticoagulants by targeting human factor XIIIa (FXIIIa). All available anticoagulants are associated with a significant risk of bleeding. Current anticoagulants inhibit directly or indirectly thrombin and/or factor Xa. This is the reason why they are clinically effective, but it is also the reason why they cause bleeding. The central hypothesis is that inhibiting FXIIIa will result in effective protection against VTE without causing significant bleeding. In contrast to all other clotting factors which are serine proteases, FXIIIa is a transglutaminase that catalyzes the last step in the coagulation process. This unique biochemical aspect of FXIIIa has been under investigation in the context of VTE. In vitro experiments showed that treating normal human blood with an experimental transglutaminase inhibitor increases RBC extrusion from contracting clots and reduces clot size. Various studies also suggested that a certain FXIIIa polymorphism provides significant protection against VTE and that heterozygous FXIII-deficient mice do not show signs of excessive bleeding. Thus, FXIIIa may serve as a potential therapeutic target to develop a new effective treatment for VTE that does not significantly increase the bleeding risk. Despite this promise, very few FXIIIa inhibitors have been developed, all of which lack substantial selectivity as they can also inhibit other transglutaminases by blocking their active sites. Thus, I have proposed sulfonated non-saccharide glycos- aminoglycan mimetics as a platform to develop FXIIIa inhibitors. The sulfonated molecules are to inhibit FXIIIa potently and selectively through allosteric modulation. In preliminary studies, I discovered two sulfonated molecules that inhibit FXIIIa with low micromolar potencies. The two molecules inhibited FXIIIa-mediated polymerization of fibrin. The two molecules did not affect other clotting factors and did not affect the viability of three cell lines. Molecular modeling projected a plausible binding site for these molecules on FXIIIa. In this proposal, I specifically aim at using a multidisciplinary approach to establish the principles of effective and selective inhibition of FXIIIa by sulfonated molecules. I will synthesize advanced libraries of two “lead” molecules and evaluate their biochemical and biological potential as anticoagulants. The proposal is innovative because i) it puts forward a novel approach to overcome the limitations of current VTE treatment; ii) it exploits a multidisciplinary approach to investigate the specific aims; and iii) it introduces new technologies with proprietary structural and mechanistic aspects. The project is also significant because it will: i) identify 2-3 potent, specific, and allosteric FXIIIa inhibitors for future evaluation in animal models of VTE and bleeding; ii) offer new tools to better understand FXIIIa role in the coagulation physiology and pathology; iii) investigate an alternative approach to modulate FXIIIa via allostery to pave the way to transforming anticoagulants.