A molecular understanding of the coagulation cascade is key to more effectively addressing the public health burden represented by thrombotic disorders and hemophilia. Thrombosis is a leading cause of morbidity and mortality, respon- sible for approximately 10 million deaths per year worldwide and ?2 million venous thromboembolism events per year in the United States alone. The cellular membrane is central to the clotting cascade, as it provides a platform for nearly all coagulation reactions. Speci?c anionic phospholipids play a complex role in regulating the cascade through atomic-level interactions with coagulation factor membrane anchors. Closely related membrane anchors show markedly differen- tial binding and speci?city to anionic lipids, phenomena yet to be adequately explained owing to a lack of detailed structural information. There is also a dearth of atomic-level structural information regarding the membrane-bound macromolecular complexes vital to spurring clot formation as a result of the membrane dependence of coagulation complex formation. The objectives of this application are to elucidate the pivotal role of anionic phospholipid speci?city and membrane binding af?nity in regulation of coagulation (Aim 1), and to develop the ?rst complete structural model of a ternary coagulation complex integrating all available experimental information and taking the role of the membrane into account (Aim 2).
In Aim 1, membrane-bound models of GLA domains, membrane anchors common to vitamin K-dependent coagulation factors, will be developed in lipid compositions of interest using an accelerated membrane representation to capture spontaneous membrane binding and to achieve enhance sampling of protein-lipid interac- tions. Advanced free energy calculations will then be performed to determine GLA domain membrane binding af?nity.
In Aim 2, protein-protein docked structures of the extrinsic ternary complex will ?rst be developed incorporating all avail- able experimental information. These initial approximate structures will then be used to determine collective variables, or measures of degree of complex formation, along which to apply force in nonequilibrium (biased) molecular dynamics simulations of ternary complex formation. The nonequilibrium simulations will be performed on a phospholipid bilayer to fully take into account the effects of membrane interactions. The results of the computational studies in Aims 1 and 2 will be used to identify key protein-protein and protein-lipid interactions and these interactions will be further examined using experimental mutagenesis studies. The knowledge gained through this work has potential to allow development of novel therapies with targeted speci?city, such as thrombotic inhibitors targeting speci?c GLA domains and recombinant mutant coagulation factors of increased potency for treating hemophilia.
Despite the fact that blood clotting system disorders are a leading cause of morbidity and mortality in the United States, our understanding of blood clotting reactions at a molecular level remains abysmally incomplete. The proposed studies will shed new light on the regulation of the blood clotting cascade, with particular focus on acquiring a detailed, mechanistic understanding of how and why blood clotting reactions, including formation of protein-protein complexes, respond preferentially to speci?c lipid types. Elucidating the mechanisms by which clotting factors bind the membrane and form protein complexes will enable us to develop rational approaches to novel therapeutics targeting hematological emergencies.
| Muller, M P; Wang, Y; Morrissey, J H et al. (2017) Lipid specificity of the membrane binding domain of coagulation factor X. J Thromb Haemost 15:2005-2016 |
