Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Rojin Park; Lifang Ping; Jaewoo Song; Sung-Yu Hong; Tae-Youn Choi; Jong-Rak Choi; Oleg V. Gorkun; Susan T. Lord

doi:10.1160/TH11-10-0731

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2012; 107(05): 875-883
DOI: 10.1160/TH11-10-0731

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

Autor*innen

Rojin Park

¹Department of Laboratory Medicine, Soon Chun Hyang University Hospital, Seoul, Republic of Korea

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Lifang Ping

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Jaewoo Song

³Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
Sung-Yu Hong

⁴Cardiovascular Product Evaluation Center, Yonsei University Health System, Seoul, Republic of Korea
Tae-Youn Choi

¹Department of Laboratory Medicine, Soon Chun Hyang University Hospital, Seoul, Republic of Korea
Jong-Rak Choi

³Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
Oleg V. Gorkun

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Susan T. Lord

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Abstract

Summary

The fibrinogen γ-module has several important sites relating to fibri-nogen function, which include the high affinity calcium binding site, hole ‘a’ that binds with knob ‘A’, and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower V_max than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plas-min-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and ‘A-a’ interactions.

Keywords

Dysfibrinogenemia - γAla341 - γ-module - calcium binding - GPRP binding - ‘A-a’ interaction

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Referenzen

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