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.

Studies on the basis for the properties of fibrin produced from fibrinogen-containing γ′ chains

“…[50][51][52] GPRphoresis uses electrophoresis to cause the countermigration of Gly-pro-arg-pro amide (GPRP-NH 2 ; Bachem Biosciences, King of Prussia, PA) in GPRP-NH 2 -impregnated gels to separate GPRP-NH 2 -insoluble fibrin monomers lacking both FPAs (␣-fibrin) from a fibrin intermediate termed ␣-profibrin lacking only one FPA, and it is soluble in buffer solutions lacking GPRP-NH 2 . After electrophoresis, the band containing ␣-profibrin comigrates with fibrinogen and is distinguished from it by immunoprobing the gel with an anti-fibrin ␣17-23 antibody (Roche Diagnostics, Basel, Switzerland) that reacts with ␣-fibrin or ␣-profibin but not with fibrinogen.…”

A Mitogenic Action for Fibrinogen Mediated through Intercellular Adhesion Molecule-1

“…Fibrin-free fibrinogen (18) was provided by Dr. J. Shainoff (Cleveland Clinic Foundation, Cleveland, OH). Human fibronectin purified according to the method of Vuento and Vaheri (19) was a gift from Dr. T. Ugarova (Cleveland Clinic Foundation, Cleveland, OH).…”

“…Using electrophoretic conditions that allow the separation of fibrin from fibrinogen monomer and subsequent visualization using Coomassie violet R-250 (18), the isolated material was estimated to comprise greater than 95% fibrinogen. Preparations of Fg were also analyzed for the presence of free fibrinopeptides A and B by elution on a Sep-Pak C 18 high pressure liquid chromatography column using standard preparations of each of the fibrinopeptides (Sigma). At protein concentrations of at least 50-fold greater than those used in these experiments, amounts of fibrinopeptides A and B were below detectable levels.…”

Allosteric Effects Potentiating the Release of the Second Fibrinopeptide A from Fibrinogen by Thrombin

“…The release of a second set of fibrinopeptides, FPB, trails the release of FPA and enables further coupling of interactions within the protofibrils, which in turn enhances their lateral aggregation (9,10). Our preceding study (5) shows that the maximal level of ␣-profibrin achieved in the course of fibrin formation was half of what would be expected if the rate constants for the release of the first and second FPA were equal. The low level of ␣-profibrin suggested that either 1) there was a partial coupling of the release of the second FPA with the first or 2) ␣-profibrin was a better substrate enabling thrombin to release the remaining FPA at a rate appearing three times faster than release of the first.…”

“…The release of the first FPA produces an intermediate, ␣-profibrin, that aggregates so weakly that it had been difficult to distinguish from fibrinogen until recently (5). The release of the second FPA enables cooperative interactions of the resulting ␣-fibrin molecules with other fibrin(ogen) molecules forming either soluble double-stranded protofibrils or laterally coalesced insoluble fibrin strands.…”