The long-term goal of this research is to understand the three natural mechanisms which control the activity of thrombin, the final protease of the blood coagulation cascade: 1) prevention of thrombin formation through inhibition of upstream coagulation factor Xa;2) direct inhibition of thrombin by serpins antithrombin (AT) and heparin cofactor II (HCII);and, 3) attenuation of thrombin's procoagulant activity through cofactor binding and allosteric modulation. This will be achieved by completing the following five specific aims (SA): SA1-To determine the crystal structures of the Michaelis complexes between AT and factor Xa will define the molecular basis of AT recognition of factor Xa, the protease responsible for thrombin formation. We will solve the crystal structures of the Michaelis complexes between AT and factor Xa in the absence and presence of heparin by creating variants of AT and factor Xa designed to improve crystallization or to increase the stability of the complex to improve the likelihood of success. SA2-To determine the heparin binding mechanism of AT addresses fundamental questions concerning the basis of heparin binding specificity of AT, by determining: a) how AT interacts with low-affinity heparin;b) high resolution crystal structures of AT bound to low and high- affinity heparin and pentasaccharides;c) crystal structures of monomeric native and activated AT;d) the role of hinge region expulsion in heparin binding;and, e) the role of electrostatics in propagating the conformational change. SA3-To determine the structures of native and oligosaccharide-activated HCII will define how heparin activates thrombin inhibition by HCII. Successful completion of this aim will elucidate the molecular mechanism of HCII activation, and will aid in the development of a novel class of anti-thrombin agents HCII agonists. SA4-To determine the molecular basis of thrombin allostery investigates how thrombin activity can be altered by conformational changes induced by binding to TM or to the monovalent cation Na+, by determining the crystallographic structures of TM-bound (active site-free) thrombin, and the Na+-free ('slow') form of thrombin. Structures will be validated in solution using 2D NMR techniques. The combined crystallographic and solution NMR approach will establish, once-and-for-all, the molecular basis of thrombin allostery. SA5-To determine the Na+ binding properties of thrombin investigates the potential relevance of Na+-allostery, which depends on the affinity of thrombin for Na+ under physiological conditions. Titration studies will accurately determine the effect of temperature, ionic strength, ion type, pH, and albumin concentration on the apparent Kd of thrombin for Na+, in order to establish the potential relevance of Na+ allostery in blood coagulation. Successful completion of these five aims will significantly improve our understanding of the natural mechanisms which limit the activity of thrombin, and could lead to the development of novel therapeutic approaches for the prevention and treatment of thrombosis-a leading cause of morbidity and death in the United States, and the rest of the developed world.
Showing the most recent 10 out of 22 publications
