ISOLATION AND CHARACTERIZATION OF THE FIBRIN INTERMEDIATE ARISING FROM CLEAVAGE OF ONE FIBRINOPEPTIDE-A FROM FIBRINOGEN

The thrombin-catalyzed cleavage of N-terminal fibrinopeptide A (FPA) f rom the two A alpha-chains of fibrinogen exposes aggregation sites wit h the critical sequence GPR located just behind FPA. It is well known that exposure of both GPR sites transforms fibrinogen into self-aggreg ating, fully coagulable alpha-fibrin monomers, but the fibrin precurso r with one site exposed and one FPA intact has eluded description. The formation of this ''alpha-profibrin'' in the course of thrombin react ions and its distribution among both the aggregating and non-aggregati ng components of the reactions are characterized here by immunoprobing electrophoretic and gel chromatographic separations using monoclonal antibodies specific for FPA and for exposed GPR sites. These analyses show alpha-profibrin to be a non-aggregating derivative indistinguisha ble from fibrinogen in solutions that are rich in fibrinogen relative to dissolved fibrin. But alpha-profibrin forms soluble complexes with alpha-fibrin monomer under conditions in which it and fibrin predomina te over fibrinogen. It was isolated as a complex with fibrin by gel ch romatography of cryoprecipitates and then separated from the fibrin ei ther by electrophoretic gel shifts induced with a peptide analog of th e GPR aggregation site or by chromatographic gel shifts induced with m onoclonal anti-FPA antibody. The weak aggregation of alpha-profibrin w ith itself and with fibrinogen conforms with prior indications that co upled interactions through the paired GPR sites on fibrin monomers are pivotal to their aggregation. It is suggested that alpha-profibrin ma y be a hypercoagulable fibrin precursor because it is converted to alp ha-fibrin monomer faster than fibrinogen converts to monomer.