Hemostasis is maintained through a delicate balance of two cascades leading to the formation (coagulation) and dissolution (fibrinolysis) of a clot. The coagulation cascade is propagated through formation of three successive enzyme-cofactor complexes that rapidly leads to generation of large quantities of thrombin. The prothrombinase complex, formed between a noncovalent association between the enzyme factor Xa and cofactor Va, serves as the final generator of thrombin. Without this complex physiologically relevant levels of thrombin cannot be formed in a sufficient amount of time to form a clot before hemorrhaging occurs. While the kinetics of the prothrombinase complex have been greatly detailed, the mechanism of association between factors Xa and Va that leads to large modulations in enzymatic activity is not. Analysis of the residues at the enzyme-cofactor interface and complex structure is necessary to understand the mechanism of interaction.
The aims of this proposal are to: (1) determine the molecular mechanism of the noncovalent interactions, both within the cofactor as well as between the cofactor Va and enzyme factor Xa, required for formation of a fully active prothrombinase complex;and (2) determine the x-ray crystallographic structure of the prothrombinase complex. A recent crystal structure of inactivated factor Va has identified potential regions of cofactor stabilization and led to several computer models proposing regions of interaction at the enzyme-cofactor interface. Mutations of implicated residues involved in cofactor stabilization and enzyme- cofactor interactions will be analyzed for their effect on prothrombinase activity and regulation. Coordinated mutations on both enzyme and cofactor will identify the noncovalent interactions found at the interface and role in thrombin generation. The x-ray structure of the prothrombinase complex has the most potential for confirming residues at the interface as well as identifying additional residues and interactions. A biochemical and structural understanding of involved proteins and complexes will enable targeted drug design to assist in maintaining hemostasis. Pharmacological intervention and regulation of the coagulation cascade is a clinical necessity with more than 40% of the U.S. population suffering from some form of cardiovascular disease.
| Bravo, Maria Cristina; Orfeo, Thomas; Mann, Kenneth G et al. (2012) Modeling of human factor Va inactivation by activated protein C. BMC Syst Biol 6:45 |
| Mason, Anne B; Judson, Gregory L; Bravo, Maria Cristina et al. (2008) Evolution reversed: the ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis. Biochemistry 47:9847-55 |
