Fibrinogen residue gamma Ala341 is necessary for calcium binding and 'A-a' interactions

Abstract

The fibrinogen gamma-module has several important sites relating to fibrinogen function, which include the high affinity calcium binding site, hole 'a' that binds with knob 'A', and the D:D interface. Residue gamma Ala341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (gamma Ala341Asp), Tolaga Bay (gamma Ala341Val), and Lyon III (gamma Ala341Thr). In order to investigate the impaired polymerisation of fibrinogens gamma Ala341Asp and gamma Ala341Val to understand the role of gamma Ala341 in fibrin polymerisation and fibrinogen synthesis, we have expressed gamma Ala341Asp and gamma Ala341Val 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 SOS-PAGE showed that factor XIIIa-catalysed gamma and alpha chain cross-linking was delayed, and plasmin-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 gamma Ala341 is important for the proper conformation of the gamma-module, maintaining calcium-binding site and 'A-a' interactions.