Characterization of the molecular basis of labile heme as a prothrombotic modulator under hemolytic conditions

金属卟啉血红素是一种结合在卟啉环系统中的铁离子配位复合物，广泛分布于机体内，是血红蛋白尤其是运氧蛋白血红蛋白的重要组成部分。不平衡的血红素稳态导致血红素的释放，其清除通常由包括血红素在内的几种蛋白质调节。然而，血管内血红素水平的升高与血栓形成前、细胞毒性、促炎和补体激活效应有关，这是在患有溶血性疾病（如镰状细胞病和输血相关性溶血）的患者中观察到的有害并发症的特征。考虑到血栓的高患病率和相关的死亡率，令人惊讶的是，不稳定血红素和血栓前作用之间的机制联系尚未在分子水平上完全阐明。在此背景下最相关的蛋白质，即血液凝固系统的组成部分，不同的蛋白质，其中因子VIII，活化蛋白C和纤维蛋白原，已被确定为血红素结合甚至血红素调节的蛋白质，而其他蛋白质（凝血酶，FXIIIa）不结合血红素。关于血红素与大多数凝血因子的潜在相互作用的许多问题仍然是开放的。因此，本提案旨在通过生物物理（如UV/Vis， SPR）和结构分析（rRaman）方法阐明血红素与凝血因子FVIII和血管性血友病因子的瞬态相互作用。此外，生化和血液学功能分析以及具有高生理学相关性的细胞方法（例如，血小板/内皮细胞功能分析）将被执行。所获得的结果将被纳入血红素的机理知识图HemeKG，以交联已知的作用，并揭示迄今为止血红素的凝血调节作用的未知方面。综上所述，血红素的促凝作用将在不同的水平上，即分子、生化、生物信息学和细胞水平上应用各种光谱方法和功能分析。这将允许更详细的表征血红素的血栓形成的性质，并为溶血相关血栓的治疗提供新的视角。

The metalloporphyrin heme is a coordination complex of an iron ion incorporated in a porphyrin ring system, which is widely distributed in the body as an essential part of hemoproteins, especially the oxygen-transport protein hemoglobin. An unbalanced heme homeostasis leads to the release of heme, whose clearance is usually regulated by several proteins including hemopexin. Increased intravascular heme levels, however, have been associated with prothrombotic, cytotoxic, proinflammatory, and complement-activating effects, which characterize the harmful complications observed in patients suffering from hemolytic disorders, such as sickle cell disease and transfusion-related hemolysis. Considering the high prevalence of thrombosis and the associated mortality it is surprising that the mechanistic link between labile heme and prothrombotic effects is not yet completely elucidated on the molecular level. Concerning the most relevant proteins in this context, namely the components of the blood coagulation system, distinct proteins, among them factor VIII, activated protein C, and fibrinogen, have been identified as heme-binding or even heme-regulated proteins, while others (thrombin, FXIIIa) do not bind heme. Many questions regarding a potential interaction of heme with most of the coagulation factors are still open. This proposal thus aims at the elucidation of the transient interaction of heme with the coagulation factor FVIII and von Willebrand factor by means of biophysical (e.g., UV/Vis, SPR) and structural analysis (rRaman) methods. In addition, biochemical and hematological functional assays as well as cellular approaches with high physiological relevance (e.g., platelet/endothelial cell function analysis) will be performed. The results obtained will be incorporated into the mechanistic knowledge graph HemeKG to crosslink the already known effects and to unravel so far unknown aspects of heme’s coagulation-modulating effects. In conclusion, the procoagulant effects of heme will be targeted on different levels, i.e. the molecular, biochemical, bioinformatic, and cellular level applying a variety of spectroscopic methods and functional assays. This will allow for a more detailed characterization of heme’s prothrombotic nature and provide new perspectives for the treatment of hemolysis-associated thrombosis.