title: Isolation and characterization of the fibrin intermediate arising from cleavage of one fibrinopeptide A from fibrinogen.
abstract: The thrombin-catalyzed cleavage of N-terminal fibrinopeptide A (FPA) from the two Aalpha-chains of fibrinogen exposes aggregation sites with the critical sequence GPR located just behind FPA. It is well known that exposure of both GPR sites transforms fibrinogen into self-aggregating, fully coagulable alpha-fibrin monomers, but the fibrin precursor with one site exposed and one FPA intact has eluded description. The formation of this "alpha-profibrin" in the course of thrombin reactions and its distribution among both the aggregating and non-aggregating 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 indistinguishable 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 predominate over fibrinogen. It was isolated as a complex with fibrin by gel chromatography of cryoprecipitates and then separated from the fibrin either by electrophoretic gel shifts induced with a peptide analog of the GPR aggregation site or by chromatographic gel shifts induced with monoclonal anti-FPA antibody. The weak aggregation of alpha-profibrin with itself and with fibrinogen conforms with prior indications that coupled interactions through the paired GPR sites on fibrin monomers are pivotal to their aggregation. It is suggested that alpha-profibrin may be a hypercoagulable fibrin precursor because it is converted to alpha-fibrin monomer faster than fibrinogen converts to monomer.
authors.affiliation: Department of Cell Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Shainoff
authors.forename: G B
authors.initials: GB
authors.lastname: Smejkal
authors.forename: P M
authors.initials: PM
authors.lastname: DiBello
authors.forename: O V
authors.initials: OV
authors.lastname: Mitkevich
authors.forename: P J
authors.initials: PJ
authors.lastname: Levy
authors.forename: C E
authors.initials: CE
authors.lastname: Dempfle
authors.forename: H
authors.initials: H
authors.lastname: Lill
mesh_terms: D000595:Amino Acid Sequence; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry*; D005344:Fibrinopeptide A / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D009842:Oligopeptides; D005344:Fibrinopeptide A; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin