Thrombin Hydrolysis of V29F and V34L Mutants of Factor XIII (28−41) Reveals Roles of the P₉ and P₄ 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.

Factor XIII A-Subunit V34L Variant Affects Thrombus Cross-Linking in a Murine Model of Thrombosis

“…17 Changes in the peptide sequence at positions V29F and V34L affected the interaction with thrombin, with residue 34 playing a more important role than residue 29. 18 The importance of residue 34 was further highlighted by a V34A substitution showing a decrease in k cat /K m , indicating a decrease in hydrolysis by thrombin. 19 Our new data, using fulllength rhFXIII-A 2 protein, support some of these earlier findings based on synthetic peptides.…”

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

“…The Leu variant of the Val34Leu polymorphism has been shown to favor the interaction with thrombin leading to improved activation kinetics. 20 The loop as well as the 4 N-terminal amino acids are flexible and hence not resolved in the crystal structure. Otherwise, the AP-FXIII assumes a defined conformation within the FXIII A-subunit dimer.…”

Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms

“…116 A recent study by Trumbo and Maurer 117 on peptide (FXIII A-subunit 28-41) hydrolysis and conformation 29, indicating that the main interaction between thrombin and factor XIII resides in the P4-P1 (Val/Leu34-Arg37) segment of the activation peptide. 117 In the presence of polymerizing fibrin, the catalytic efficiencies of thrombin cleavage of the activation peptide are increased approximately 10-fold, but activation of factor XIII Leu34 remains faster than that of Val34, with a catalytic efficiency of 4.8 compared with 2.2 M Ϫ1 sec Ϫ1 . 112 An interesting observation is that release of the Leu34 activation peptide proceeds at a similar rate to that of fibrinopeptide A, whereas the rate of Val34 peptide cleavage is in tandem with that of fibrinopeptide B.…”