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Thromb Haemost 2013; 109(02): 199-206
DOI: 10.1160/TH12-08-0609
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

An engineered fibrinogen variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links

Rojin Park
1   Department of Laboratory Medicine, Soon Chun Hyang University Hospital, Seoul, Republic of Korea
2   Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
,
Lifang Ping
2   Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
,
Jaewoo Song
3   Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
,
Joo-Young Seo
4   Department of Family Medicine, Soon Chun Hyang University Hospital, Seoul, Republic of Korea
,
Tae-Youn Choi
1   Department of Laboratory Medicine, Soon Chun Hyang University Hospital, Seoul, Republic of Korea
,
Jong-Rak Choi
3   Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
,
Oleg V. Gorkun
2   Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
,
Susan T. Lord
2   Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
› Author Affiliations Financial support:This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2007–331-E00205 to R.P.) and the grant from the NIH/NHLBI (HL031048 to S.T.L.).
Further Information

Publication History

Received: 25 August 2012

Accepted after major revision: 22 October 2012

Publication Date:
29 November 2017 (online)

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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
 

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