Difference in electrophoretic mobility and plasmic digestion profile between four recombinant fibrinogens, γ14;308K, γ308I, γ308A, and wild type (γ308N)

We have produced recombinant γ‐chain variant fibrinogens, γ308K, γ308I, and γ308A simultaneously with wild‐type fibrinogen, γ308N, by genetic protein engineering using Chinese hamster ovary cells. Although all three variant fibrinogens are a result of a single amino acid substitution, the aberrant γ‐chains of γ308K and γ308I fibrinogens migrated faster than γ308N. Furthermore, plasmic digestion profiles were examined in the presence of 5 m M ethylene glycol‐bis(β‐aminoethyl ether)‐ N,N,N′,N′ ‐tetraacetic acid (EGTA) or 1 m M CaCl 2 . In the presence of EGTA, the three variant fibrinogens were digested into D 1 and D 2 fragments slightly faster than wild type. In addition, the D 2 fragment derived from γ308K was further digested into D 3 by plasmin much faster than that from γ308N. These data suggest that cleavage of γ356Lys‐γ357Ala bond by plasmin in γ308K, γ308I, and γ308A is slightly accelerated and the γ302Lys‐γ303Phe bond is cleaved by plasmin rapidly in only the γ308K variant. Furthermore, the substitution of Lys for γ308Asn results in the generation of a new plasmin cleavage site between γ308Lys and γ309Gly in the presence of EGTA. In conclusion, a substitution at residue γ308Asn may cause a conformational change in the γ‐chain of fragment D affecting polymerization and plasmin cleavage.