Program Director/Principal Investigator (Last, First, Middle): KrJShnaSWamy, Sriram PROJECT SUMMARY(See instructions): The serine proteinase factor Xa (FXa) and the cofactor factor Va (FVa) are essential components of the enzymatic complex prothrombinase, the only known physiological activator of prothrombin. A major factor by which prothrombinase achieves its remarkable specificity is through exosites interactions between enzyme, substrate and cofactor. Many of these binding sites are not functional on the precursor of these proteins and the mechanism by which they are conformationally activated is not well understood. Additionally, there are still major gaps in the current understanding of how these sites are allosterically modulated and to what extent these interactions contribute to the enhanced function of the enzyme complex. The broad long-term objective of this work is to decipher these molecular processes and provide detailed mechanistic insight into FXa and FVa function within prothrombinase. In the first aim, we will investigate the role of the FX zymogen to protease transition pathway in macromolecular binding site expression within the catalytic domain. We hypothesize that intramolecular activation of FX following conformational activation of the protease plays a major role in the exposure of substrate, cofactor, and inhibitor binding sites. In the second aim, we will exploit unique FVa variants to examine prothrombin binding and examine the role this interaction plays in prothrombinase function. We hypothesize that specific acidic sequences within the heavy chain of FVa are important for thrombin binding but do not play a major role in prothrombin recognition. In the final aim, we will use FXa "zymogen-like" variants which can be thermodynamicallyrescued by FVa to examine whether they provide effective hemostasis using in vitro and in vivo models of hemophilia. We hypothesize that these variants will be resistant to protease inhibitors and be long lived in the circulation but promote thrombin generation in the presence of FVa. We will test whether these derivatives will be useful therapeutic procoagulant bypass agents in hemophilic animals. Understanding the molecular events leading to the expression of structural determinants on FXa and FVa as well as how these sites influence each other may prove useful in developing pharmacological agents directed at regulating thrombin generation.

Public Health Relevance

The proteins and cells that form a blood clot must remain in a quiescent state and only become activated following injury. This proposal seeks to better understand how one of these proteins, factor X is converted to the active form FXa. The mechanism underlying this process has relevance for better understanding and possibly treating the pathological states of hemophilia and thrombosis. PROJECT/

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL074124-10
Application #
8450263
Study Section
Special Emphasis Panel (ZHL1-PPG-J)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
10
Fiscal Year
2013
Total Cost
$361,063
Indirect Cost
$141,572
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Hanby, Hayley A; Zheng, X Long (2014) Current status in diagnosis and treatment of hereditary thrombotic thrombocytopenic purpura. Hereditary Genet 3:
Ivanciu, Lacramioara; Krishnaswamy, Sriram; Camire, Rodney M (2014) New insights into the spatiotemporal localization of prothrombinase in vivo. Blood 124:1705-14
Bao, Jialing; Xiao, Juan; Mao, Yingying et al. (2014) Carboxyl terminus of ADAMTS13 directly inhibits platelet aggregation and ultra large von Willebrand factor string formation under flow in a free-thiol-dependent manner. Arterioscler Thromb Vasc Biol 34:397-407
Kowalska, M Anna; Zhao, Guohua; Zhai, Li et al. (2014) Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation. Arterioscler Thromb Vasc Biol 34:120-6
Krishnaswamy, S (2013) The transition of prothrombin to thrombin. J Thromb Haemost 11 Suppl 1:265-76
Bennett, Charles L; Jacob, Sony; Dunn, Brianne L et al. (2013) Ticlopidine-associated ADAMTS13 activity deficient thrombotic thrombocytopenic purpura in 22 persons in Japan: a report from the Southern Network on Adverse Reactions (SONAR). Br J Haematol 161:896-8
Zheng, X L (2013) Structure-function and regulation of ADAMTS-13 protease. J Thromb Haemost 11 Suppl 1:11-23
Reshetnyak, Andrey V; Nelson, Bryce; Shi, Xiarong et al. (2013) Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region. Proc Natl Acad Sci U S A 110:17832-7
Bunce, Matthew W; Bos, Mettine H A; Krishnaswamy, Sriram et al. (2013) Restoring the procofactor state of factor Va-like variants by complementation with B-domain peptides. J Biol Chem 288:30151-60
Thalji, Nabil; Camire, Rodney M (2013) Parahemophilia: new insights into factor v deficiency. Semin Thromb Hemost 39:607-12

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