In blood coagulation, the enzyme thrombin cleaves fibrinogen, sites for fibrin polymerization are revealed, and fibrin clot formation begins. Activated Factor XIII, a transglutaminase, is responsible for catalyzing the formation of covalent crosslinks between fibrin molecules and in fibrin-enzyme complexes. Thrombin activates Factor XIII by cleaving a peptide sequence that lies across the Factor XIII catalytic site. The thrust of this research project will be to examine in solution the structural features that govern the activation and substrate specificity of Factor XIII.
The specific aims are designed to prove the following hypotheses: 1) There is no obvious consensus sequence for defining the substrate specificity of Factor XIII for glutamine donating groups. The glutamine containing substrates are hypothesized to interact with the Factor XIII enzyme surface using similar bound conformations with defined flexible and/or solvent exposed regions. Peptides based on natural and synthetic substrates will be examined. 2) The Factor XIII activation peptide segment has components that resemble the fibrinogen Aalpha chain, the thrombin receptor, and PPACK, all of which interact with thrombin. The Factor XIII activation peptide segment is hypothesized to adopt a bound conformation that allows for the most effective interactions with the thrombin surface. An understanding of how unique amino acid positions within this segment participate in binding may lead to the design of Factor XIII enzymes whose degree of activation and optimal target site can be controlled. A model system for these studies is Factor XIII 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 address these specific aims, a combination of kinetic studies, hydrogen/deuterium exchange followed by MALDI-TOF mass spectrometry, 1D proton line broadening NMR, 2D TOCSY, 2D transferred NOESY, and molecular modeling will be used. Understanding the molecular details of how Factor XIII is activated and how its substrate specificity is regulated is critical considering the role this enzyme plays in increasing the risk of heart disease, stroke, and arteriosclerosis.
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