title: Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation.
abstract: In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation.
authors.affiliation: Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, USA.
authors.forename: Toni A
authors.initials: TA
authors.lastname: Trumbo
authors.forename: Muriel C
authors.initials: MC
authors.lastname: Maurer
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D005176:Factor XIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D005176:Factor XIII; D013917:Thrombin