The aim of this study was to investigate the interactions of t-PA and
plasminogen with
fibrin derived from an abnormal
fibrinogen detected in a 40-year-old male patient who had had an episode of
thrombophlebitis with
pulmonary embolism. An abnormal
fibrinogen was diagnosed on the basis of prolonged
thrombin and reptilase times also detected in two other family members.
Fibrinogen purified from plasma, in the presence of
protease inhibitors, by
glycine precipitations, gel filtration and affinity chromatography, was devoid of
plasminogen,
fibronectin, and vWf. SDS-PAGE analysis according to Laemmli under reducing conditions, showed an abnormal gamma chain (approximately 50% of the total) migrating in a more anodic position (M(r) 48 kDa). By PCR amplification and
DNA sequencing, the abnormality was identified as an Asn308-->Lys mutation of the gamma chain. Since such a mutation constitutes a new
plasmin cleavage site as first reported for
fibrinogen Kyoto I, it may modify interactions of
plasminogen and t-PA with carboxy-terminal
lysine residues.
Ligand-binding studies were therefore performed using intact and
plasmin-degraded
fibrin surfaces obtained from the abnormal
fibrinogen. The
plasminogen and t-PA binding isotherms obtained with the abnormal
fibrinogen were similar to the control. Moreover, the stimulation by
fibrin of
plasminogen activation by t-PA was not different from the control. These results suggest (i) that the
lysine 308 residue may not be exposed to
plasmin cleavage in
fibrin, and (ii) that the thrombotic accident of the propositus cannot be explained by an abnormality of the
plasminogen/t-PA binding to
fibrin.