Summary
A fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular
ε -lysyl-γ -glutamyl covalent cross-links between α chains to form α polymers and
between γ chains to form γ dimers. In a previous study we characterised fibrinogen
Seoul II, a heterozygous dysfibrinogen in which a cross-linking acceptor site in Aα
chain, Gln328, was replaced with Pro (AαQ328P). Following on the previous study, we
investigated whether the alteration of Gln residues Aα328 and Aα366 affects fibrin
polymerisation and α chain cross-linking. We have expressed three recombinant fibrinogens:
AαQ328P, AαQ366P, and AαQ328,366P in Chinese hamster ovary cells, purified these fibrinogens
from the culture media and performed biochemical tests to see how the introduced changes
affect fibrin polymerisation and α chain cross-linking. Thrombin-catalysed fibrin
polymerisation of all variants was impaired with the double mutation being the most
impaired. In contrast, sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
immunoblot analysis showed α polymer formation with all three engineered proteins.
This study demonstrates that AαQ328 and AαQ366 are important for normal fibrin clot
formation and in the absence of residues AαQ328 and AαQ366, other Gln residues in
the a chain can support FXIIIa-catalysed fibrin cross-linking.
Keywords
Fibrin alpha chain - cross-linking - Glutamine acceptor - AαQ328 - AαQ366
