Abstract
Essentials The roles of β‐barrels 1 and 2 in factor XIII (FXIII) are currently unknown.FXIII truncations lacking β‐barrel 2, both β‐barrels, or full length FXIII, were made.Removing β‐barrel 2 caused total loss of activity, removing both β‐barrels returned 30% activity.β‐barrel 2 is necessary for exposure of the active site cysteine during activation. SummaryBackgroundFactor XIII is composed of an activation peptide segment, a β‐sandwich domain, a catalytic core, and, finally, β‐barrels 1 and 2. FXIII is activated following cleavage of its A‐subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis.ObjectivesTo assess the functional roles of β‐barrels 1 and 2 in FXIII, we expressed and characterized the full‐length FXIII A‐subunit (FXIII‐A) and variants truncated to residue 628 (truncated to β‐barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to β‐sandwich).MethodsProteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system.Results and ConclusionsCircular dichroism spectroscopy and tryptophan fluorometry indicated that full‐length FXIII‐A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of β‐barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of β‐barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full‐length FXIII‐A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the β‐barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the β‐barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual β‐barrel domains in modulating access to the FXIII active site region.
Highlights
In the final step of the blood coagulation cascade, fibrin monomers polymerise to generate a fibrin clot
To assess the functional roles of -barrels 1 and 2 in Factor XIII (FXIII), we expressed and characterised the full-length FXIII A subunit (FXIII-A) subunit (FXIII-A) and variants truncated to residue 628 [truncated to β-barrel 1 (TB1)], 515 [truncated to catalytic core (TCC)] and 184 [truncated to -sandwich (TBS)]
Circular dichroism spectroscopy and tryptophan fluorometry indicate that full length FXIII-A and the truncations TCC and TB1 retain their secondary and tertiary structure
Summary
In the final step of the blood coagulation cascade, fibrin monomers polymerise to generate a fibrin clot. FXIIIa is capable of cross-linking other substrates into the fibrin clot network whose functions include inhibition of fibrinolysis (e.g. α2-antiplasmin [2, 3]), increased thrombin generation at the clot surface (e.g. factor V [4, 5]), and platelet adhesion to the clot (e.g. collagen [6, 7]). The A-subunits are folded into four distinct domains, from N- to C-terminus: the activation peptide, β-sandwich, catalytic core (containing the active site), β-barrel 1 and β-barrel 2 domains [8] (Figure 1B). Thrombin cleaves the activation peptide from the N-terminus of each A-subunit monomer and, in the presence of calcium, the B-subunits of FXIII dissociate from the A-subunits exposing the active sites of the A-subunits in the catalytic core to substrates [11,12,13]
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