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

Abstract

<jats:title>Summary</jats:title><jats:p>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 Vmax 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.</jats:p>

Combined Congenital Hypodysfibrinogemia and Factor XI Deficiency in a Chinese Family

“…Hypodysfibrinogenemia has been described in Fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr) [19-21]. Fibrinogen residue γAla341 is necessary for calcium binding and “A-a” interactions [22]. Therefore, the probable reason for the association of a genetic defect in the γHis340Arg family with hypodysfibrinogenemia may be an altered clot structure due to perturbed fibrin polymerization.…”

Evaluation and Improvement of in vitro Detection Methods of Thrombin Inhibitor Activity

“…Therefore, the reason for the difference in absorbance is the solidification of the solution leading to an increase in turbidity rather than a change in color. However, absorbance of 350 nm or 650 nm were chosen in the similar methods [8,9]. Secondly, there is no direct relationship between thrombin inhibitory activity and the value of absorbance.…”