Biochimica et Biophysica Acta (BBA) - Protein Structure
Plasmic degradation of human fibrinogen I. Structural characterization of degradation products
Abstract
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1. Fragments X, Y, D, and E were prepared by plasmic degradation of human fibrinogen and purified by gel filtration and chromatography. Molecular weights of reduced polypeptide chains of fibrinogen and its degradation products were studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea.
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2. Plasmic hydrolysis of fibrinogen was interrupted at different digestion stages, and the release of polypeptides from fibrinogen was studied by polyacrylamide electrophoresis of non-reduced and reduced incubation mixtures.
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3. In the early phase of plasmic hydrolysis, the β and γ chains of fibrinogen lose small molecular weight peptides at the N-terminal and C-terminal ends, respectively. Large polypeptides are progressively being split from the C-terminal part of the α chain. Fragment X was found to be a heterogenous population of molecules differing in the length of their α chains.
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4. On further digestion, one pair of disulfide-bound β and γ chains is released from the fragment X, giving rise to Fragment Y, which in turn splits into another β-γ dimer and Fragment E. Our results suggest that the β-γ dimer corresponds to Fragment D and confirm the partial identity of Fragment E with the disulfide knot.
References (18)
- P.A. McKee et al.
Arch. Biochem. Biophys.
(1966) - V.J. Marder et al.
J. Biol. Chem.
(1969) - D. Mills et al.
Biochem. Biophys. Res. Commun.
(1970) - P.J. Gaffney et al.
FEBS Lett.
(1971) - A.Z. Budzyński et al.
Biochim. Biophys. Acta
(1967) - K. Weber et al.
J. Biol. Chem.
(1969) - M.C. Brummel et al.
Anal. Biochem.
(1970) - G.A. Dudek et al.
Biochim. Biophys. Acta
(1970) - D.A. Mills et al.
Arch. Biochem. Biophys.
(1969)
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Fibrinogen catabolism within the procoagulant VX-2 tumor of rabbit lung in vivo: Effluxing fibrin(ogen) fragments contain antiangiogenic activity
2004, Journal of Laboratory and Clinical MedicineMany types of solid tumors are known to be procoagulant environments. This is partly because a hyperpermeable vascular system within the tumor allows plasma hemostatic factors to accumulate in relatively high concentrations in the stroma, and many solid-tumor cells express tissue factor or a procoagulant factor. These circumstances appear to exist in the VX-2 lung tumor of the New Zealand White (NZW) rabbit, and they sustain a measurable turnover of stromal deposits of fibrin(ogen). We have measured the turnover of fibrinogen within tumors of the VX-2 tumor–burdened rabbit and analysed the catabolic products of fibrin(ogen) and the status of fibrinolysis in tumor-derived interpleural effusate. Using intravenously injected 125I-labeled rabbit fibrinogen as a marker, we found that fibrinogen (approximate blood concentration 1740 μg/mL) passed from blood to VX-2 tumor stroma, saturating the tumor at a concentration of approximately 348 μg fibrinogen/g in approximately 12 hours. We measured fibrin(ogen) fragments, at a concentration of approximately 292 μg/mL, in interpleural effusates that we recovered from 13% of the VX-2–burdened rabbits. Unreduced fibrin(ogen) fragments consisted of 4 major components with a relative molecular mass of approximately 250,000 (assumed to be fragment X; approximately 9% of total fragments from densitometry of immunoblots), 200,000 (d-dimer; 41%), 110,000 (fragment D; 49%), and 50,000 to 55,000 (fragment E; 1%–2%) kD. Total fibrin(ogen) fragments immunopurified from effusates exhibited an antiangiogenic effect when subjected to a chick embryo chorioallantoic membrane procedure. Interpleural effusates were devoid of plasmin activity or active plasminogen activator inhibitor-1 but contained plasmin complexes and active urokinase-like plasminogen activator (uPA), α2-antiplasmin, and thrombin-activatable fibrinolysis inhibitor. We speculate that VX-2 cells release uPA to activate fibrinolysis within the tumor stroma. Catabolic products of hemostasis (eg, fibrinolytic fragments, angiostatin) flux from the stroma into the interpleural space, thereby providing a net antiangiogenic property to the effusate and ultimately to the lymphatic and circulatory systems.
Contact with the N termini in the central E domain enhances the reactivities of the distal D domains of fibrin to factor XIII<inf>a</inf>
1995, Journal of Biological ChemistryThe reaction of Factor XIIIa with fibrin is the last enzyme-catalyzed step on the coagulation cascade, leading to the formation of a normal blood clot. The finding that fibrin is preferred by the cross-linking enzyme about 10-fold over the circulating fibrinogen suggests the operation of a unique substrate-level control for the orderly functioning of the physiological process in the forward direction. An important task is to elucidate the molecular mechanism for the transmission of the signal generated by the thrombin-catalyzed cleavage in the central E domain of fibrin to the distant Factor XIIIa-reactive glutamine residues. By focusing on the substrate sites present in γ chain remnants of D type domains of fibrinogen and by employing the approach of fragment complementation with the regulatory E domain, which represents the thrombin-modified portion of fibrin, we have now succeeded in reconstructing in solution the phenomenon of kinetic enhancement for the reaction with Factor XIIIa.
Two D type preparations (truncated fibrinogen, ~250 kDa and D‘, ~105 kDa) were obtained by digestion of human fibrinogen with endo Lys-C. Neither product could be cross-linked by Factor XIIIa, but as shown by the incorporation of dansylcadaverine, both were acceptor substrates for the enzyme. The plasmin-derived D (~105-kDa) product, however, could be cross-linked into DD dimers. In all cases, the admixture of E fragments exerted a remarkable boosting effect on the reactions with Factor XIIIa. Even with native fibrinogen as substrate, cross-linking of γ chains was enhanced in the presence of E. Nondenaturing electrophoresis was used to demonstrate the complex forming potential of E fragments with fibrinogen, truncated fibrinogen, D‘, or D. The GPRP tetrapeptide mimic of the GPRV N-terminal sequence of the α chains in the E fragments, abolished both complex formation and the kinetic boosting effect of E on the reactions of substrates with Factor XIIIa. Thus, the N-terminal α chain sequences seem to act as organizing templates for spatially orienting the D domains, probably during the protofibrillar assembly of the fibrin units, for favorable reaction with Factor XIIIa.
The role of the endothelium in in vivo anticoagulation
1991, Journal of Oral and Maxillofacial SurgeryEndothelial cells play an important role in the anticoagulation of circulating blood. They participate in at least four mechanisms that aid in preventing vascular occlusion by platelet plugs or fibrin deposition. These mechanisms include the fibrinolytic pathway, the enhancement of action of endogenous inhibitors of coagulation, the inhibition of cofactors required for fibrin formation, and the synthesis and release of platelet inhibitors. Thus, the endothelium can no longer be viewed merely as a passive barrier separating the blood from the interstitial matrix. Instead, it must be viewed as an active participant in the maintenance of blood fluidity and the prevention of thrombogenesis.
Involvement of the cooh-terminal portion of the alpha-chain of fibrin in the branching of fibers to form a clot
1987, Thrombosis ResearchA modified fibrinogen molecule which is missing the COOH-terminal portion of the Aα chain has been used in structural investigations of the mechanism of assembly of the fibrin clot. Brief plasmin digestion of human fibrinogen, followed by ammonium sulfate fractionation and column chromatography, yielded a highly clottable fragment X-like preparation. Molecules in this preparation contain mostly intact Bβ and γ chains, but are missing the COOH-terminal two-thirds of the Aα chain. Clots formed by addition of thrombin to this fragment were mechanically unstable and easily dispersed. Electron microscopy showed that the clots consist mainly of a suspension of individual fibers, in contrast to clots made from native fibrinogen, which are highly branched. It appears, therefore, that a part of the COOH-terminal two-thirds of the α chain is necessary for branching of fibers to form a stable three-dimensional gel. Intermolecular interactions of this portion of the α chain are consistent with certain of its unusual features, such as its apparent existence, in part, as a single polypeptide chain and its involvement in Factor XIIIa-mediated ligation between molecules.
Measurement in human blood of fibrinogen/fibrin fragments containing the bβ 15-42 sequence
1982, Thrombosis ResearchThis paper describes a radioimmunoassay for the Bβ 15–42 peptide derived from human fibrinogen or fibrin. Iodinated Bβ 15–42 is bound by specific antiserum and binding can be completely inhibited by excess of the non-iodinated Bβ 15–42, Bβ 1–42 or Bβ 1–118. These peptides cannot be distinguished in this assay. Furthermore, fibrinogen can also completely inhibit binding of iodinated Bβ 15–42 peptide. Due to the cross-reaction with fibrinogen, clinical blood samples require a processing step prior to their use in this radio-immunoassay. Ethanol precipitation allows for fibrinogen removal and a near quantitative recovery of both added Bβ 15–42 peptide as well as of the endogenous blood peptide(s) containing the Bβ 15–42 sequence. The mean level of Bβ 15–42 immunoreactive material in normal individuals was found to be 0.41 pmol/ml while that in one group of patients was 20–40 times this value.
Factors influencing the structure of terminal plasmin degradation products of human fibrinogen and fibrin
1981, BBA - Protein StructureExperiments have been carried out with fibrinogen and with purified degradation products of fibrinogen and fibrin which demonstrate that the structure of D fragments obtained after prolonged plasmin digestion is influenced by several factors in the media.
The previously described protective effect of calcium ions on the γ-chain carboxy-terminals of fibrinogen against attack has been confirmed by working at high plasmin concentrations and/or in the presence of 2 M urea.
Several compounds such as EDTA, EGTA, citrate and iminodiacetic acid appear to have a separate effect. In the absence of calcium ions these compounds appear to make the γ-chain carboxy-terminal ends of the D and D-dimer fragments more susceptible to plasmin digestion.
Finally, as demonstrated by experiments with purified D-E complexes from fibrinogen and with whole fibrinogen digests, the E moiety of the D-E complexes appears to be capable of protecting the D moiety against low plasmin concentrations also in the absence of calcium ions.