THE MUTATION IN FIBRINOGEN BICETRE-II (GAMMA-ASN308-]LYS) DOES NOT AFFECT THE BINDING OF T-PA AND PLASMINOGEN TO FIBRIN

The aim of this study was to investigate the interactions of t-PA and plasminogen with fibrin derived from an abnormal fibrinogen detected i n a 40-year-old male patient who had had an episode of thrombophlebiti s 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) migra ting in a more anodic position (M(r) 48 kDa). By PCR amplification and DNA sequencing, the abnormality was identified as an Asn308-->Lys mut ation of the gamma chain. Since such a mutation constitutes a new plas min cleavage site as first reported for fibrinogen Kyoto I, it may mod ify 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 fibri nogen. The plasminogen and t-PA binding isotherms obtained with the ab normal fibrinogen were similar to the control. Moreover, the stimulati on 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 throm botic accident of the propositus cannot be explained by an abnormality of the plasminogen/t-PA binding to fibrin.