Degradation Products Of Human Fibrinogen Research Articles

Clinical significance of peripheral blood UL16 and DR-70 for the early diagnosis and prognostic evaluation of colorectal cancer.

Early diagnosis of colorectal cancer (CRC) is of great significance to improve the survival rate and quality of life of patients, but early diagnosis of CRC requires more sensitive techniques. Peripheral blood UL16-binding protein 2 (ULBP2) and human fibrinogen degradation products (DR-70) are the main indicators for the diagnosis of malignant tumors. To assess ULBP2 and DR-70 potential for the early diagnosis and prognostic evaluation of CRC to provide a reference. This study involved 60 patients with early-stage CRC (CRC group), 50 patients with benign colorectal tumors (benign group), and 50 healthy patients (control group) enrolled at the Affiliated Hospital of Jiangnan University and Jiangsu Province Official Hospital between January, 2020 and January, 2022. ULBP2 and DR-70 levels in the blood were determined and differences among the three groups and early diagnostic values for CRC were determined. Patients with CRC were divided into the good prognosis and poor prognosis groups, and ULBP2 and DR-70 levels in the blood and diagnostic values were compared. ULBP2 and DR-70 serum levels were significantly higher in the CRC group than in the control and benign groups (P < 0.05); however, no significant differences were observed between the benign and control groups (P > 0.05). Among the 60 patients with CRC followed up for two years, two died (3.33%) and 15 exhibited tumor metastasis, progression, or recurrence (25.00%). ULBP2 and DR-70 serum levels were significantly higher in the poor prognosis group than in the good prognosis group (P < 0.05). A receiver operating characteristic curve was plotted. Area under the curve, sensitivity, and specificity of serum ULBP2 with DR-70 for the early diagnosis of CRC were higher than those of the single serum indices (P < 0.05) in both the good and poor prognosis groups. ULBP2 and DR-70 serum levels were significantly high in patients with early-stage CRC. They improved the diagnostic rate of early-stage CRC and predicted patient prognosis, thereby showing clinical application potential.

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation.

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.

Factors influencing the structure of terminal plasmin degradation products of human fibrinogen and fibrin

Experiments 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.

Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen

Fibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observations of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction. These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.

Anticoagulant peptides obtained from human fibrinogen degradation products by plasmin

Anticoagulant peptides obtained from human fibrinogen degradation products by plasmin have been studied. These peptides caused a siginificant prolongation of the plasma prothrombin time and partial thromboplastin time of normal plasma. Moreover, they showed an inhibitory effect on factors VIII, IX, XI, and XII in the intrinsic blood coagulation system. It is suggested that they might have feed-back inhibition against these factors and control thromboplastogenesis in blood coagulation, especially in the intrinsic clotting mechanism.

Radioimmunoassay of fibrinogen-fibrin degradation products: Assay for fragment E-related neoantigen -methodological aspects

E fragment and its antigenically-related degradation products of human fibrinogen (Fg) and/or fibrin (Fb) have been measured in both normal and pathological plasmas by a specific radioimmunoassay (RIA) for the neoantigenic expression of E(Eneo) which is engendered after the plasmic cleavage of Fg or Fb. High titer antisera were produced in rabbits against Fg-E (E derived from Fg). The anti-Fg-E sera harvested were absorbed with a cyanogen bromide-activated immunoadsorbent column coupled with intact Fg. The Fg-absorbed antisera were employed to develop a double antibody RIA which detected < 1 ng/ml of Fg-E and exhibited little cross-reactivity of Fg at 1 mg/ml. This Eneo RIA did not discriminate between Fg-E and Fb-E (E derived from Fb) in the competitive inhibition profile, and hence it measured total Eneo antigens. The displacement curves of normal plasmas generally showed a parallel relationship to the standard which permitted the calculation of normal plasma Eneo immunoreactivity (ca. 15 ng/ml). Considerably higher plasma Eneo immunoreactivities have been observed in patients with consumption coagulopathy. Modifications of the Eneo RIA which permit the attainment of this sensitivity are described.

Effects of proteolytic degradation products of human fibrinogen and of human factor VIII on platelet aggregation and vascular permeability

Products of proteolysis of highly purified preparations of human fibrinogen (FDP) and of human factor VIII (VIII-DP) by plasmin were compared with respect to their ability to inhibit platelet aggregation and to enhance the permeability of rat skin microvasculature. Low molecular weight dialysable FDP (LMW-FDP, m. w. under 2754) induced complete inhibition of platelet aggregation in PRP at concentrations of and above 2. 5 mg/ml. Slight or very pronounced increase in skin microvasculature permeability was observed at LMW-FDP doses of 10 μg and 78 μg, respectively. In contrast, the whole digest of factor VIII as well as LMW-VIII-DP appeared inactive. Unfractionated digests of fibrinogen and fibrin formed either by thrombin of Defibrase exhibited the same antiaggregating potency.

A Simple and Practical Method for Isolation of an Early Plasmin Degradation Product of Human Fibrinogen

One of the earliest plasmin degradation products of human fibrinogen, so-called fragment A, was isolated by a simple method. This peptide has a molecular weight of approximately 22,500, migrating electrophoretically at beta-area, and its amino acid composition shows a very high content of glycine, serine, threonine and proline, and a markedly low content of hydrophobic amino acids. This fragment does not react against anti-fibrinogen; however, the anti-serum of this fragment reacts strongly with fibrinogen.

The Half-Life of Plasmic Degradation Products of Human Fibrinogen in Rabbits

Human fibrinogen and purified plasmic degradation fragments X (stages 1 and 2), D and E were labelled with 125-I using the lactoperoxidase method. The chromatographic, electrophoretic and immunologic properties of the labelled proteins were found to be similar to those of non-labelled fragments. All the degradation products diffused rapidly (T 1/2 0.27-0.75 hours) from the intravascular space of rabbits, as compared with fibrinogen (4.26 hours). In addition, the metabolic half-life was found to be 49.3 hours for fibrinogen, as compared with only 5.6, 6.1, 2.3 and 1.4 for fragments X (stage 1), X (stage 2), D and E, respectively. The metabolic half-life roughly reflects the molecular size of the degradation products.

Structure of Plasmic Degradation Products of Human Fibrinogen: FIBRINOPEPTIDE AND POLYPEPTIDE CHAIN ANALYSIS

Direct determination of fibrinopeptides A and B in purified plasmic degradation products of human fibrinogen shows that fibrinogen and Fragments X and Y contain 2 moles of fibrinopeptide A per mole; Fragment E has approximately 0.3 mole of fibrinopeptide A per mole. Fibrinopeptide B is present in only trace amount in some Fragment X preparations and is absent from Fragments Y and E. Fragment D contains neither fibrinopeptide A nor B. These data and the results on molecular weight, carbohydrate content, and reactivity with thrombin of polypeptide chains of the purified degradation products were used to assign these chains to the Aα, Bβ, or γ chains of the original fibrinogen molecule. Fragment X has altered Aα chains with a molecular weight of 25,000, Bβ chains that are either intact (58,000) or cleaved at the NH2-terminal end (53,000), and γ chains that are either intact (48,000) or cleaved at the COOH-terminal end (40,000). Fragment Y has two distinct sets of polypeptide chains; the heavier ones are almost the same as those of the smallest Fragment X species. The lighter chains of Fragment Y are similar to those of Fragment E, which has three major components of 13,000, 8,000, and 10,000 assigned tentatively as Aα, Bβ, and γ fibrinogen chain remnants, respectively. The chains of Fragment D have been classified as a 13,000 molecular weight α chain remnant, a 42,000 β chain remnant and a heterogeneous group (39,000, 32,000, and 26,000) of γ chain remnants. The presence of 2 moles of fibrinopeptide A per mole of Fragment Y and the demonstration of the heavy and light sets of polypeptide chains in Fragment Y provide substantive evidence for its asymmetric structure.

Plasmic degradation products of human fibrinogen II. Chemical and immunological relation between fragment E and N-DSK

The relation between Fragment E and N-DSK was studied by immunological and chemical techniques. Fragment E was shown to have antigenic determinants in common with N-DSK. Cleavage of Fragment E with cyanogen bromide produced one main fragment (“E-knot”), reacting with anti N-DSK serum and having a molecular weight of 43 000. On thrombic digestion this fragment, like Fragment E, released Fibrinopeptide A. “E-knot” had the same content of disulphide bridges and neutral sugars as Fragment E and N-DSK. Tryptic peptides of “E-knot” were separated by two dimensional thin layer electrophoresis-chromatography. Subsequently the peptides were subjected to amino acid analysis and the amino acid compositions compared with those of tryptic peptides of N-DSK. Our results show that “E-knot” is part of N-DSK and most molecules satisfy the formula: (A α 1 Ala-51Met, β54Lys-115Met, γ1 Tyr-58Lys) 2. Some molecules appear to be lacking one or more of the following structural features: Aα1Ala-16Arg, Aα51Met, β54Lys, β54Lys-58Lys and γ54Thr-58Lys. In addition to these specified structures Fragment E contains sequences extending beyond Aα51Met and Bβ115Met.

Plasmic degradation products of human fibrinogen. I. Isolation and characterization of fragments E and D and their relation to “disulfide knots”

Separation of degradation products from human fibrinogen (Fragments A–E) after digestion with plasmin has been performed, using gel filtration in 10 % acetic acid on Sephadex G-100. Fragments D and E have been partly characterized and related to “disulfide knots” obtained from cyanogen bromide (CNBr) treated fibrinogen. Fragment E seems to contain three pairs of polypeptide chains, linked together by disulfide bonds, forming a dimeric molecule with a molecular weight of about 50 000. Immunologically and chemically it was found to have similarities with the NH 2-terminal disulfide knot (N-DSK). Fragment D was found to be heterogeneous with a molecular weight range of 86 000 – 96 000. It seems to be built up by three polypeptide chains linked by disulfide bridges. Two or three hydrophobic disulfide containing products can be obtained from Fragment D after cleavage of this with CNBr. These products are similar with the three hydrophobic disulfide knots (H1-DSK, H2-DSK and H3-DSK), isolated from native fibrinogen after cleavage with CNBr. Furthermore our results suggest that Fragment D is located in a close proximity to N-DSK.