The specific proteolytic activation steps of blood coagulation are catalysed by serine proteinases that are homologous to each other and to the archetypic serine proteinases of digestion. In many cases, explanations for the molecular basis of the distinctive macromolecular substrate specificity of the coagulation reactions remain speculative and incomplete. Evidence suggests that prothrombin activation by the prothrombinase complex, results from interactions at an extended macromolecular recognition site (Exosites) on the enzyme followed by active site interactions prior to cleavage and product release. We will use prothrombin activation catalysed by the prothrombinase complex as a paradigm for specific macromolecular substrate recognition and cleavage by coagulation complexes to investigate the basis for its substrate specificity and function. Using prethrombin 2 as a substrate analog, binding and stopped flow kinetic measurements will be used to provide a complete kinetic and thermodynamic description of the stepwise interactions that lead to substrate recognition and cleavage. We will test the hypothesis that exosite, rather than active site interactions, determine substrate affinity and binding specificity for the macromolecular substrate. The significance of these findings towards prothrombinase function will be tested in studies of prothrombin and meizothrombin cleavage. By comparing the function of factor Xa with prothrombinase, we will determine if modulation of binding interactions can explain the increased Vmax for the reaction which accompanies prothrombinase assembly. Recombinant activation site mutants of the substrate will be used to determine if binding specificity determined by exosite interactions directs cleavage at the scissile bond. By the use of recombinant mutants of factor Xa, we will test the hypothesis that sites removed from the catalytic site of the protease are responsible for determining substrate binding specificity. We will also extend approaches developed in studies with prothrombinase to other coagulation reactions to determine if exosite-dependent substrate recognition is a prevalent mechanism through which coagulation proteinases achieve their specificity. We believe that the approaches contained in this proposal will provide unanticipated insights into the function of the coagulation enzymes. The delineation of the determinants of macromolecular substrate specificity will likely suggest novel approaches for therapeutic targeting of these reactions in thrombotic and vascular disease states.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062523-04
Application #
6537577
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
1999-04-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
4
Fiscal Year
2002
Total Cost
$443,093
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Cao, Wenjing; Krishnaswamy, Sriram; Camire, Rodney M et al. (2008) Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13. Proc Natl Acad Sci U S A 105:7416-21
Krishnaswamy, S (2005) Exosite-driven substrate specificity and function in coagulation. J Thromb Haemost 3:54-67
Schmidt, Amy E; Stewart, Jonathan E; Mathur, Akash et al. (2005) Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding. J Mol Biol 350:78-91
Orcutt, Steven J; Krishnaswamy, Sriram (2004) Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin. J Biol Chem 279:54927-36
Boskovic, Danilo S; Troxler, Thomas; Krishnaswamy, Sriram (2004) Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study. J Biol Chem 279:20786-93
Lu, Genmin; Broze Jr, George J; Krishnaswamy, Sriram (2004) Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III. J Biol Chem 279:17241-9
Orcutt, Steven J; Pietropaolo, Concetta; Krishnaswamy, Sriram (2002) Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate. J Biol Chem 277:46191-6
Wilkens, Matthias; Krishnaswamy, Sriram (2002) The contribution of factor Xa to exosite-dependent substrate recognition by prothrombinase. J Biol Chem 277:9366-74
Buddai, Sai K; Toulokhonova, Larisa; Bergum, Peter W et al. (2002) Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase. J Biol Chem 277:26689-98
Boskovic, D S; Krishnaswamy, S (2000) Exosite binding tethers the macromolecular substrate to the prothrombinase complex and directs cleavage at two spatially distinct sites. J Biol Chem 275:38561-70

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