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

Abstract

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, ␣-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of ␣-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na ؉ binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of ␣-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified ␣-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na ؉ . The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na ؉ , whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.Fibrin formation by thrombin depends on the cleavage of the N-terminal fibrinopeptides A (FPA) 1 from the two A␣-chains of fibrinogen (1-4). 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. The interactions between monomers involve 1) the neo-N-terminal GPR domain unmasked by the FPA release from the central E-domain (6) and 2) the complementary sites in the two D-domains at opposite ends of the trinodular (D-E-D) shaped molecules (7,8). 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 th...

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

“…This method requires the presence of GPRP-NH 2 in the thrombin-containing reaction mixture. 52 We found that the overall rate of fibrinopeptide release was inversely proportional to the concentration of GPRP-NH 2 , and its presence slowed the rate of peptide release from both fibrinogens. We carried out several ␣-profibrin generation experiments at concentrations of GPRP-NH 2 ranging from 4 to 12 mM, and in every case we observed that ␣-profibrin generation was slower from fibrinogen 2 than from fibrinogen 1.…”

“…[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.…”

“…Integration of the curves for each fibrinogen indicated that the overall amount of ␣-profibrin generated from each fibrinogen was similar (4.30 ϫ 10 9 counts for peak 1 vs 4.27 ϫ 10 9 counts for peak 2), a finding in conformance with previous comparisons of fibrinogen 2 and unchromatographed fibrinogen. 52 …”

Cross-linking of Wild-type and Mutant α2-Antiplasmins to Fibrin by Activated Factor XIII and by a Tissue Transglutaminase

“…Tissue transglutaminase (tTG), a single-chain enzyme that is widely distributed in cells and tissues, does not require activation by proteolysis. The substrate specificity of FXIIIa for ␤-casein, fibrin/fibrinogen, or their peptide derivatives differs from that of tTG (7)(8)(9). Among the plasma proteins that have Gln substrate site(s) for FXIIIa, ␣ 2 AP is recognized as having the highest kinetic efficiency (k cat /K m(app) ), with its substrate site being the second Gln from the N terminus (10,11).…”

Transglutaminase-catalyzed crosslinking of the Aα and γ constituent chains in fibrinogen

“…Under forced conditions, however, similar ␥⅐␥ and A␣ n crosslinking can also be achieved with fibrinogen. In contrast, TG reacts quite readily with fibrinogen in the soluble phase, generating A␣ p ⅐␥ q type of crosslinked, intermolecular chain combinations (8,9) as well as A␣⅐␥ crosslinking within the monomeric fibrinogen units:…”