Detection and characterization of molecular interfaces of FXIII-Fibrinogen complex by employing integrative hybrid approaches

凝血途径的内在和外在途径最终汇聚为一条共同途径，该途径涉及血浆转谷氨酰胺酶因子XIII （FXIII）对预形成的纤维蛋白凝块的共价交联的强大作用，赋予其机械强度。血凝块是由纤维蛋白纤维、沉淀的血小板、红细胞、补体和炎症成分组成的大量物质，使其成为一种凝聚的物质，进一步通过团块形成和伤口愈合来停止出血，这是激活炎症的结果。凝血因子XIII不仅负责凝聚纤维蛋白团块，而且由于其转谷氨酰胺酶活性，还将各种血浆成分拴在凝块上。对这些分子的概观及其在血浆中的作用和行为直接表明，纤维蛋白的前体纤维蛋白原是血栓形成的直接效应蛋白，而FXIII是其直接调节剂。在血浆中，存在促酶FXIII与纤维蛋白原的相互作用，但这种相互作用的确切位置、残基、系结姿势和有效强度尚不清楚。鉴于这些酶原在血浆中大量存在，有报道表明它们与血清白蛋白直接结合，这可能是其在血浆中寿命长的原因。在目前的提案中，这是我上一个DFG拨款的延伸，我想提出一个尝试，以表征FXIII和纤维蛋白原在其天然酶原状态下的相互作用界面，如在血浆中所见。这将有助于fxiii -纤维蛋白原复合物的结构评估。作为提案的一部分，我还将尝试并部分成功地解决天然因子XIII- A2B2复合物的结构，这在蛋白质数据库中尚未全部可用。了解这些蛋白在血浆中的结构相互作用行为将有助于了解它们在损伤时释放活性纤维蛋白和FXIII-A2的激活模式。通过结构研究将揭示两种分子中作为相互作用界面的表面斑块，绘制与FXIII和纤维蛋白原相关的未解凝血病变，将指导我们了解它们，从而改进管理和护理。

The intrinsic and extrinsic pathways of coagulation pathway converge terminally to a common pathway that involves strong role of plasma transglutaminase Factor XIII (FXIII), towards covalent crosslinking of pre-formed fibrin clots, imparting it mechanical strength. A blood-clot is a massive mass of fibrin fibers, precipitating platelets, RBCs, complement, and inflammatory components making it a condensed mass of material that further ensues cessation of bleeding by clump formation and wound healing as a result of activated inflammation. Coagulation Factor XIII is responsible for not just condensing this fibrin mass, but also tethering diverse plasma components onto the clot because of its transglutaminase activity. A conspectus view of these molecules, their roles and behavior in plasma directly indicates that fibrinogen, the precursor of Fibrin acts as a direct effector protein for clot formation whereas FXIII is its direct modifier. In plasma, there exists an interaction of zymogenic FXIII with fibrinogen, however the exact positions, residues, tethering poses, and the effective strength of this interaction is unknown. Given that these zymogens are abundantly present in plasma there have been reports indicating their direct binding to serum albumin which is likely responsible towards its longevity in plasma. In the present proposal, which is an extension of my last DFG grant, I would like to put forward an attempt to characterize the interaction interfaces among FXIII and Fibrinogen in their native zymogenic states as seen in plasma. This will aide in structural assessment of FXIII-Fibrinogen complex. As a part of the proposal, I would also attempt and I am partially successful in resolving the structure of the native Factor XIII- A2B2 complex, which is not available in totality as yet in the protein databases. Understanding the structural interaction behavior of these proteins in plasma would develop insights towards activation modes of both of them that releases active Fibrin and FXIII-A2 for their action, upon injury. This through structural investigation would reveal surface patches in both the molecules that act as interaction interfaces, mapping under-resolved coagulopathies related to FXIII and Fibrinogen will guide us towards understanding them, and hence towards an improved management and care.