Perturbations in Factor XIII Resulting from Activation and Inhibition Examined by Solution Based Methods and Detected by MALDI-TOF MS

Abstract

Factor XIII can be activated proteolytically by thrombin cleavage of the activation peptide or non-proteolytically by exposure to 50 mM Ca2+. The resultant transglutaminase cross-links Q and K residues within the noncovalently associated fibrin clot. Hydrogen deuterium exchange coupled with MALDI-TOF MS demonstrated that FXIII activation protects regions within the beta sandwich (98-104) and the beta barrel 1 (526-546) from deuterium, while exposing the potential Q substrate recognition site (220-230) to deuteration (Turner, B. T., Jr., and Maurer, M. C. (2002) Biochemistry 41, 7947-7954). Chemical modification indicated the availability of several residues upon activation including K73, K221, C314, and C409 (Turner, B. T., Jr., Sabo, T. M., Wilding, D., and Maurer, M. C. (2004) Biochemistry 43, 9755-9765). In the current work, activations of FXIII by IIa and by Ca2+ as well as FXIIIa inhibition by the K9 DON peptide (with the Q isostere 6-diazo-5-oxo-norleucine) and iodoacetamide were further examined. New findings unique for FXIIIaIIa included alkylation of C238 and C327, acetylation of K68, and increased proteolysis of 207-214. By contrast, FXIIIaCa led to increased proteolysis of 73-85 and 104-125 and to a loss of K129 acetylation. The FXIIIa inhibitors K9 DON and iodoacetamide both promoted even greater protection from deuteration for the beta sandwich (98-104) and beta barrel 1 (526-546). Interestingly, only K9 DON was able to block modification of catalytic core C409 near the dimer interface. The solution based approaches reveal that activation and inhibition lead to local and long range effects to FXIII(a) and that many are influenced by Ca2+ binding. Important glimpses are being provided on FXIIIa allostery and the presence of putative FXIIIa exosites.

Interactions between factor XIII and the αC region of fibrinogen

“…The low-affinity interaction was dependent on calcium, which induces a conformational change in the ␤-barrel 1 and the ␤-sandwich domains of thrombin-cleaved FXIII-A. [13][14][15] In addition, calcium could mediate the interaction with Glu396. However, the lowaffinity interaction was independent of catalytic activity, showing that it did not arise from a transient covalent intermediate.…”

“…Furthermore, it was observed that the ␣C region 233-425 does not bind to rFXIII-A activated with thrombin only but also required the presence of calcium that induces a conformational change that exposes portions of FXIII-A that are not structurally exposed in the nonactivated form [13][14][15] ( Figure 5B). Binding did not depend on catalytic activity because blocking the rFXIII-A active site Cys314 with iodoacetamide did not inhibit binding to the ␣C region 233-425, which occurred to the same degree as activated rFXIII-A in the absence of iodoacetamide ( Figure 5C).…”

“…[10][11][12] Calcium has been shown to cause small but significant conformational changes in FXIII-A during activation, exposing potential exosites within FXIII-A. [13][14][15] Activated FXIII-A 2 stabilizes the forming protofibril by introducing ⑀-amino(␥-Glutamyl)Lysine cross-links between carboxyl terminal portions of adjacent fibrin ␥ chains, before lateral association of the protofibril. 16 Cleavage of FpB and subsequent release of the ␣C regions from the central E region initiates lateral aggregation of protofibrils and enables activated FXIII-A 2 to cross-link adjacent ␣C, stabilizing the developing fiber and making it more resistant to fibrinolysis.…”

Perturbations in protein dynamics associated with ligand binding to the blood coagulation enzymes thrombin and Factor XIII.

“…In addition to probing FXIIIa inhibition, new insight has also been obtained regarding the conformational features associated with the activation of FXIIIa by Ca 2+ and by IIa, thus augmenting our previous investigations (174,175). The current studies thus provide valuable information about events occurring to the β sandwich, the activation peptide, the dimer interface, the catalytic core, and β barrels 1 and 2 (178). (269).…”

“…FXIIIa has now been developed that incorporates the Q isostere 6-diazo-5-oxo-norleucine into the FXIIIa substrate peptide K9. The focus of the work has proceeded to observe in solution the conformational changes associated with inhibition by K9 DON compared to IAA (178). In addition to probing FXIIIa inhibition, new insight has also been obtained regarding the conformational features associated with the activation of FXIIIa by Ca 2+ and by IIa, thus augmenting our previous investigations (174,175).…”