Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants

Abstract

Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28′41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its Km upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins.

Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: A strategy for controlling fibrin architecture

“…NMR studies indicated that L34 and P36 promote stabilizing P 4 to P 2 interactions that better anchor the AP segment onto the thrombin active site surface and promote hydrolysis [37]. Additional kinetic studies further demonstrated the critical role that the 34 (P 4 ) position can play in controlling thrombin-catalyzed hydrolysis of the FXIII (28–41) AP segment [25, 38–40]. Results with recombinant full length FXIII variants involving these same AP residues support this proposal [41, 42].…”

“…As a result, FXIIIa catalyzed crosslinking could be initiated earlier or later in the fibrin clot assembly process. Moreover, specific FXIII mutants could be matched with thrombin species that have either procoagulant or anticoagulant properties [3, 4, 19, 40]. To design such FXIII variants, more information is needed on how the FXIII AP segment interacts with specific regions of wild-type thrombin and its potential therapeutic mutants.…”

“…Wild-type and mutant thrombin studies have already been carried out with FXIII AP (28–41) peptides containing V34, L34, P34, or F34 [36, 40]. The FXIII P34 variant allowed platelet PAR4 character to be introduced into the FXIII AP sequence (Table 1) [47].…”

“…Earlier NMR studies had revealed that the FXIII F34 provided the same beneficial P 4 -P 2 interactions with the thrombin surface as L34 [39]. Importantly, the FXIII F34 AP was demonstrated to be the first FXIII-based substrate that exhibited little change in K m upon loss of thrombin residue W215 [40]. An aromatic residue at the P 4 position was proposed to be an effective strategy for promoting FXIII activation in the presence of an anticoagulant thrombin.…”

“…Hydrolysis of FXIII W34 and Y34 APs (28–41) were examined with WT thrombin and the mutants W60dA, Y60aA, L99A, I174A, W215A, and WE. The kinetic effects were compared with FXIII V34, L34, and F34 [36, 40]. Results reveal that FXIII W34 and F34 are promising candidates for creating therapeutic FXIII – thrombin mutant systems that contain some crosslinking ability in an environment of decreased fibrin(ogen) clotting.…”