Fbg αC 389–402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions

• Published in: Biochemistry (Easton) 2023-07, Vol.62 (14), p.2170-2181
• Main Authors: Ablan, Francis D. O., Maurer, Muriel C.
• Format: Article
• Language: English
• Subjects: Factor XIII - chemistry; Factor XIII - genetics; Factor XIII - metabolism; Factor XIIIa - genetics; Factor XIIIa - metabolism; Fibrinogen - chemistry; Hydrophobic and Hydrophilic Interactions; Static Electricity
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.3c00066

Coagulation Factor XIII (FXIII) stabilizes blood clots by cross-linking glutamines and lysines 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 is a binding site for thrombin-activated FXIII, (FXIII-A*), with αC E396 promoting FXIII-A* binding and activity in αC. The current study aimed to discover additional residues within Fbg αC 389–402 that accelerate transglutaminase activity toward αC. Electrostatic αC residues (E395, E396, and D390), hydrophobic αC residues (W391 and F394), and residues αC 328–425 were studied by mutations to recombinant Fbg αC 233–425. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) cross-linking and gel-based fluorescence monodansylcadaverine (MDC) cross-linking assays. Truncation mutations 403 Stop (Fbg αC 233–402), 389 Stop (Fbg αC 233–388), and 328 Stop (Fbg αC 233–327) reduced Q237-GEE and MDC cross-linking compared to wild-type (WT). Comparable cross-linking between 389 Stop and 328 Stop showed that FXIII is mainly affected by the loss of Fbg αC 389–402. Substitution mutations E396A, D390A, W391A, and F394A decreased cross-linking 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, cross-linking was 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 key contributors in enhancing αC cross-linking.