Our research builds upon 30 years of continuously funded work on allosteric theory and experimental studies on the structure, function and regulation of trypsin-like proteases and their zymogens. Our work has led to elucidation of the allosteric nature of the trypsin fold 1,2 and construction of a thrombin mutant 3-7 that just completed Phase I (NCT03453060) for the treatment of thrombotic complications and is now moving to Phase II testing (NCT039638950). Other notable contributions include identification of the Na+ binding site of thrombin 8, the structures of thrombin bound to fragments of relevant physiological substrates 9-11, and the structures of zymogen precursors of thrombin 12,13.
We are currently pursuing three areas of investigation:
- Structure and dynamics of prothrombin (HL049413)
- Mechanism of protein C activation (HL139554)
- Molecular determinants of thrombin allostery (HL147821)
We are particularly interested in two basic interactions of the blood coagulation cascade, i.e., the interaction of prothrombin with prothrombinase leading to generation of thrombin and blood clotting, and the interaction of the thrombin-thrombomodulin complex with protein C initiating the negative feed-back loop of the coagulation response. Both of these interactions have been studied for decades by several groups but remain incompletely understood at the molecular level because of the lack of adequate structural information. Our experimental approach merges enzymology, protein engineering and X-ray structural biology with innovative techniques like smFRET, 19F NMR and cryo-EM. Our long term goal is to resolve the molecular architecture of prothrombin and protein C, free and bound to their biological activators, and to gain information on underlying dynamics, mechanism of activation and epitopes involved in the interactions.
The lab has unique expertise and resources to train students and postdocs in state-of-the-art approaches to biological interactions.
- Dang, O.D., Vindigni, A. & Di Cera, E. An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. Proc Natl Acad Sci U S A 92, 5977-81 (1995).
- Gohara, D.W. & Di Cera, E. Allostery in trypsin-like proteases suggests new therapeutic strategies. Trends Biotechnol 29, 577-585 (2011).
- Dang, Q.D., Guinto, E.R. & Di Cera, E. Rational engineering of activity and specificity in a serine protease. Nat Biotechnol 15, 146-9 (1997).
- Cantwell, A.M. & Di Cera, E. Rational design of a potent anticoagulant thrombin. J Biol Chem 275, 39827-30 (2000).
- Gruber, A. et al. Relative antithrombotic and antihemostatic effects of protein C activator versus low molecular weight heparin in primates. Blood 109, 3733-3740 (2007).
- Flick, M.J. et al. The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A. Blood 117, 6326-6337 (2011).
- Berny, M.A. et al. Thrombin mutant W215A/E217A acts as a platelet GpIb antagonist. Arterioscler Thromb Vasc Biol 18, 329-334 (2008).
- Di Cera, E. et al. The Na+ binding site of thrombin. J Biol Chem 270, 22089-92 (1995).
- Pozzi, N., Barranco-Medina, S., Chen, Z. & Di Cera, E. Exposure of R169 controls protein C activation and autoactivation. Blood 120, 664-670 (2012).
- Gandhi, P.S., Chen, Z. & Di Cera, E. Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1. J Biol Chem 285, 15393-15398 (2010).
- Bah, A., Chen, Z., Bush-Pelc, L.A., Mathews, F.S. & Di Cera, E. Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4. Proc Natl Acad Sci USA 104, 11603-11608 (2007).
- Chen, Z., Pelc, L.A. & Di Cera, E. Crystal structure of prethrombin-1. Proc Natl Acad Sci U S A 107, 19278-19283 (2010).
- Pozzi, N., Bystranowska, D., Zuo, X. & Di Cera, E. Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy. J Biol Chem 291, 18107-16 (2016).