In blood clotting, the enzyme thrombin cleaves fibrinogen, sites for fibrin polymerization are revealed, and fibrin clot formation begins. Activated Factor XIII is responsible for catalyzing the formation of covalent crosslinks between fibrin molecules and in fibrin-enzyme complexes. Factor XIII can be activated through cleavage of an activation peptide segment by thrombin or nonproteolytically in the presence of high calcium or high sodium/low calcium. The long-term objective of our research project is to examine in solution the structural features that govern the activation and substrate specificity of Factor XIII. Understanding these molecular details is critical considering the role of FXIII in increasing the risk of heart disease, stroke, and arteriosclerosis.
The specific aims are an extension of ongoing studies and will address three hypotheses: 1) The reactive glutamines of certain FXIII substrates primarily target a distinctive surface within the transglutaminase active site region. Such studies are important since the substrate specificity of FXIII is not clearly defined, 2) FXIII undergoes subtle conformational changes upon activation. The degree and surface coverage of these changes will increase following introduction of a substrate or inhibitor, and 3) The FXIII activation peptide segment utilizes key positions to promote effective interactions with a thrombin surface whose properties are regulated by individual thrombin residues. A model system for such studies is FXIII V34L, a common polymorphism that has been correlated with protection against myocardial infarction and is more susceptible to thrombin cleavage than the native sequence. To test these hypotheses, a combination of kinetic studies, hydrogen/deuterium exchange, chemical modification methods, MALDI-TOF mass spectrometry, solution NMR, and surface plasmon resonance methods will be used. Information will be obtained on the selectivity of FXIII for glutamine-containing substrates, on the solvent accessibility changes that FXIII undergoes upon activation and ligand binding, and on the kinetic/structural features of the thrombin - FXIII Activation Peptide complex. Lay Summary: Factor XIII is a key enzyme in the formation of blood clots. While very important for wound healing, the clots can also increase risk for heart disease and stroke. Research will focus on understanding how Factor XIII is turned on and how it selects its targets. Studies may lead to novel medical strategies to control the actions of Factor XIII and the resultant clot architecture.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-HEME-C (03))
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Link, Rebecca P
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University of Louisville
Schools of Arts and Sciences
United States
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Jadhav, Madhavi A; Goldsberry, Whitney N; Zink, Sara E et al. (2017) Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: A strategy for controlling fibrin architecture. Biochim Biophys Acta 1865:1246-1254
Mouapi, Kelly Njine; Bell, Jacob D; Smith, Kerrie A et al. (2016) Ranking reactive glutamines in the fibrinogen ?C region that are targeted by blood coagulant factor XIII. Blood 127:2241-8
Doiphode, Prakash G; Malovichko, Marina V; Mouapi, Kelly Njine et al. (2014) Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay. Anal Biochem 457:74-84
Malovichko, Marina V; Sabo, T Michael; Maurer, Muriel C (2013) Ligand binding to anion-binding exosites regulates conformational properties of thrombin. J Biol Chem 288:8667-78
Woofter, Ricky T; Maurer, Muriel C (2011) Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen-deuterium exchange coupled with MALDI-TOF MS. Arch Biochem Biophys 512:87-95
Jadhav, Madhavi A; Lucas, R Cory; Goldsberry, Whitney N et al. (2011) Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants. Biochim Biophys Acta 1814:1955-63
Jadhav, Madhavi A; Isetti, Giulia; Trumbo, Toni A et al. (2010) Effects of introducing fibrinogen Aalpha character into the factor XIII activation peptide segment. Biochemistry 49:2918-24
Cleary, D B; Doiphode, P G; Sabo, T M et al. (2009) A non-reactive glutamine residue of alpha2-antiplasmin promotes interactions with the factor XIII active site region. J Thromb Haemost 7:1947-9
Sabo, T Michael; Maurer, Muriel C (2009) Biophysical investigation of GpIbalpha binding to thrombin anion binding exosite II. Biochemistry 48:7110-22
Sabo, T Michael; Brasher, P Bradley; Maurer, Muriel C (2007) Perturbations in factor XIII resulting from activation and inhibition examined by solution based methods and detected by MALDI-TOF MS. Biochemistry 46:10089-101

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