During the previous funding period, we have gathered strong evidence that binding of thrombomodulin (TM) at exosite 1 on thrombin alters the active site of the enzyme towards protein C activation. Our hypothesis is that TM alters thrombin by """"""""dynamic allostery"""""""". This discovery has important implications for the development of better anticoagulants and for understanding the potential of TM fragments in the treatment of disseminated intravascular coagulation. To gain information on the dynamic allostery in the thrombin-TM interaction, we will obtain answers to the following questions:
Aim 1. How does TM alter the thermodynamic balance of ligand binding at the thrombin active site? We will use isothermal titration calorimetry (ITC) to measure how binding of TM fragments at exosite 1 alters the thermodynamic parameters of various ligands binding to the active site. Active site ligands will include therapeutic direct thrombin inhibitors.
Aim 2. What role is played by specific residues in TMEGF456t, a fully active TM fragment, in allosterically changing the activity of thrombin? Mutations will be introduced into TMEGF456t and the function of these mutants will be ascertained. Direct thrombin binding, ability to activate protein C, and ability to alter the thrombin active site will be measured. Some mutants that do not directly contact thrombin but that fail to alter the active site of thrombin will be characterized by NMR.
Aim 3. What are the critical residues in the transmission line connecting the TM binding site with the 90sCT loop at the active site of thrombin? We will make site directed mutations in thrombin along the strand that connects exosite 1 to the 90sCT loop at the active site. Each mutant will be assayed for protein C activation and subjected to amide H/D exchange experiments comparing the mutant thrombin to wild type thrombin both in the presence and absence of TM fragments.
Aim 4. What are the internal backbone dynamics of thrombin and how are they affected by TM binding? NMR relaxation experiments will be performed to measure the backbone dynamics of thrombin in the presence and absence of TM fragments. These experiments will reveal backbone dynamics changes that occur within thrombin upon TM binding that are not visible from the crystal structure.
Aim 5. Is TMEGF456t a useful anticoagulant? Can we specifically target it to platelets? In discussions with Whyte Owen (Mayo Clinic), we devised a scheme to target TMEGF456t to platelets by creating a fusion of p-Selectin glycoprotein ligand 1 (PSGL-1) toTMEGF456t. Dr. Stephen R. Hanson (Oregon Health Sciences) will test this fusion for anticoagulant activity in the baboon animal model. Mutant TMs will also be studied. These experiments will help to understand how the in vitro biophysical measurements relate to in vivo function.
Thrombin is the last enzyme in the coagulation cascade and is responsible for activating fibrinogen to make fibrin clots. Uncontrolled thrombin causes blood clots that occlude the flow of blood through blood vessels resulting in heart attacks, strokes, pulmonary emboli, and venous thrombosis. These diseases are the most common causes of mortality in the US. The project goal is to understand the thrombin-thrombomodulin interaction, which is critical for regulating the generation of thrombin. Thrombomodulin provides the key switch that shuts off production of thrombin. When thrombomodulin binds to thrombin, the complex has a new activity that shuts down further production of thrombin. Our hope is that by understanding how thrombomodulin alters the activity of thrombin, we may be able to mimic this interaction with better pharmaceuticals that control hemostasis.
|Handley, Lindsey D; Fuglestad, Brian; Stearns, Kyle et al. (2017) NMR reveals a dynamic allosteric pathway in thrombin. Sci Rep 7:39575|
|Handley, Lindsey D; Treuheit, Nicholas A; Venkatesh, Varun J et al. (2015) Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin. Biochemistry 54:6650-8|
|Boechi, Leonardo; Pierce, Levi; Komives, Elizabeth A et al. (2014) Trypsinogen activation as observed in accelerated molecular dynamics simulations. Protein Sci 23:1550-8|
|Fuglestad, Brian; Gasper, Paul M; McCammon, J Andrew et al. (2013) Correlated motions and residual frustration in thrombin. J Phys Chem B 117:12857-63|
|Fuglestad, Brian; Gasper, Paul M; Tonelli, Marco et al. (2012) The dynamic structure of thrombin in solution. Biophys J 103:79-88|
|Gasper, Paul M; Fuglestad, Brian; Komives, Elizabeth A et al. (2012) Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities. Proc Natl Acad Sci U S A 109:21216-22|
|Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A (2011) Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin. Biochemistry 50:4590-6|
|Koeppe, Julia R; Beach, Muneera A; Baerga-Ortiz, Abel et al. (2008) Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin. Biochemistry 47:10933-9|
|Truhlar, Stephanie M E; Croy, Carrie H; Torpey, Justin W et al. (2006) Solvent accessibility of protein surfaces by amide H/2H exchange MALDI-TOF mass spectrometry. J Am Soc Mass Spectrom 17:1490-7|
|Koeppe, Julia R; Komives, Elizabeth A (2006) Amide H/2H exchange reveals a mechanism of thrombin activation. Biochemistry 45:7724-32|
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