Fbg αC 389-402 enhances factor XIII crosslinking in the fibrinogen αC region via electrostatic and hydrophobic interactions.

Abstract

Coagulation Factor XIII (FXIII) hinders fibrinolysis via transglutaminase activity in fibrin and other proteins. FXIII activity in the fibrinogen αC region (Fbg αC 221-610) is critical for clot stability and growth. Fbg αC 389-402 acts as a binding site for thrombin-activated FXIII, (FXIII-A∗), with αC E396 promoting FXIII-A∗ binding and activity. The current study aimed to discover additional residues within Fbg αC 389-402 that accelerate transglutaminase activity toward αC (233-425). αC E395 and D390 were proposed to form ionic interactions with FXIII that improve binding. αC W391 and F394 were hypothesized to augment FXIII-fibrinogen αC binding through favorable hydrophobic contacts with FXIII residues. Truncated Fbg αC 328-425 was also examined for any potential impact on FXIII activity. Fbg αC D390, W391, F394, E395, E396, and residues 328-425 were studied by select mutations to recombinant Fbg αC 233-425. FXIII activity was quantified by monitoring glycine ethyl ester (GEE) crosslinking to αC Q237 over time by mass spectrometry. Truncation mutations “403 Stop” (Fbg αC 233-402), “389 Stop” (Fbg αC 233-388), and “328 Stop” (Fbg αC 233-327) experienced reduced Q237-GEE crosslinking compared to wild-type (WT). Comparable crosslinking between “389 Stop” and “328 Stop” shows that FXIII is mainly affected by the loss of Fbg αC 389-402. Substitution mutations E396A, D390A, W391A, and F394A decreased crosslinking relative to WT, whereas E395A, E395S, E395K, and E396D had no effect. Similar FXIII-A∗ activities were observed for double mutants (D390A,E396A) and (W391A,E396A), relative to D390A and W391A, respectively. In contrast, crosslinking was further reduced in (F394A,E396A), relative to F394A. In conclusion, Fbg αC 389-402 boosts FXIII activity in Fbg αC, with D390, W391, and F394 identified as additional residues that enhance αC crosslinking.