Investigating interactions between Factor XIII activation peptide segments and thrombin by kinetic and NMR methods

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 characterize activation peptide binding to thrombin. V34A and V34I were examined to explore further the role of residue 34. A Factor XIII-fibinopeptide hybrid peptide was designed to promote interactions of the FXIII peptide with thrombin more like what is observed for Fibrinogen Aα (7–16). EPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28–41), FXIII AP (28–41) V34L, FXIII AP (28–41) V341, FXIII AP (28–41) V34A, FXIII AP (28–41) V29F, FXIII AP (28–41) V34L V29F, and, FXIII AP-Aα (28–41). The V34L mutation leads to improvements in both K_m and k_cat whereas the V29F mutation primarily affects K_m. Changes to V34A and V34I lead to a reduction in K _m over the V34 peptide with no change or a reduction in k_cat. The kinetics of hydrolysis for the hybrid peptide were comparable to that of the V34L V29F peptide and Fbg Aα(7–20). Interactions of the peptides with the thrombin surface 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 Aα (7–16), thrombin receptor PARI (38–60), and Factor XIII (28–37). In solution, the 34XVPR37 segments of the Factor XIII activation peptides serve as the major anchor points onto thrombin. The side chain of L34 exhibits a more intimate interaction with P36 when bound to thrombin's apolar binding site than V34 or 134. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward 34XVPR37 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 PARI than to fibrinogen Aα. Mutations at these different sites may prove useful in controlling Factor XIII activation.