Binding Properties of Monoclonal Antibodies against Human Fragment D-Dimer of Cross-Linked Fibrin to Human Plasma Clots in an In Vivo Model in Rabbits

SummaryTwo (MA-15C5 and MA-8D3) out of approximately 500 monoclonal antibodies, obtained by fusion of P3X63-Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified fragment D-dimer from human fibrin, demonstrated a more than 1,000-fold higher affinity for fragment D-dimer than for native fibrinogen. MA-15C5 was directed against a neoantigenic determinant only expressed in fragment D-dimer. MA-8D3 reacted equally well with fragment D-dimer of crosslinked fibrin and with fragment D of non-crosslinked fibrin but not with fragment D of fibrinogen. Both monoclonal antibodies did not crossreact with rabbit fibrin and its degradation products.The binding of 125I-labeled Fab fragments to human plasma clots, introduced and aged for 1 hr in the jugular vein of heparinized rabbits was studied. Following injection of an equimolar mixture of Fab fragments derived from MA-15C5 and MA-8D3, the clot to blood ratios of radioactivity increased from 3.2 ± 1.2(mean ± SD) at 4 hr to 7.2 ± 1.4 at 17 hr. The binding of Fab fragments of MA-15C5 and MA-8D3 was independent of the age (1 to 72hrs) of the clot and of heparin anticoagulation and was only slightly decreased (by 20%) in the presence of circulating human fibrinogen (90 mg/kg body weight) and of human crosslinked fibrin degradation products at a plasma concentration of 10 pg/ml. The binding of Fab fragments of MA-15C5 and MA-8D3 to occlusive human plasma clots in the femoral artery of rabbits was comparable to that of the non-occlusive human plasma clots in the jugular vein. The Fab fragments of MA-15C5 and MA-8D3, labeled with 123I to a specific activity of 10 μCi/pg were injected intravenously (3 μg/kg) in 72 rabbits with a nonocclusive 0.2 ml human plasma clot in the jugular vein and in 7 control rabbits that underwent the surgical procedure without clot formation. Total body scans performed at hourly intervals revealed a higher relative increase in gamma counts over the thrombus region in the group with thrombus as compared to controls: at 6 hr 54 ± 18 vs 16 ± 13% (mean ± SEM, p <0.1) and at12 hrs 35 ± 11 vs –7 ± 12 (p <0.05). The vein segment to blood ratios of 123I at 24 hrs were 6.6 ± 2.4 in the group with clot and 1.5 to 0.7 in the control group (p <0.01). We conclude that these Fab fragments may have a sufficiently high fibrin-affinity to allow in vivo thrombus localization by external scanning.

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THROMBUS IMAGING WITH MURINE MONOCLONAL ANTIBODIES AGAINST FIBRIN FRAGMENT D-DIMER IN A RABBIT JUGULAR VEIN THROMBOSIS MODEL

10.1055/s-0038-1642891 ◽

1987 ◽

Author(s):

P Holvoet ◽

J M Stassen ◽

D Collen

Keyword(s):

Monoclonal Antibodies ◽

Jugular Vein ◽

Myeloma Cell ◽

Jugular Vein Thrombosis ◽

Line Production ◽

Fab Fragments ◽

Thrombosis Model ◽

Murine Monoclonal Antibodies ◽

D Dimer

Three murine monoclonal antibodies (MA-6C1, MA-8D3 and MA-15C5) reacting with fragment D-dimer from crosslinked fibrin but not with monomeric fragment D were obtained by immunization of Balb/c mice with the highly purified fragment, fusion of spleen cells with a myeloma cell line, production of. ascites fluid in mice and purification of the antibodies on Affigel Blue. Fab fragments were isolated from papain digests. The IgG and Fab fragments were labeled with 125I, 131I or 123I using lactoper-oxidase. The disposition rates (t1/2) and thrombus to blood ratios, measured in groups of 3 rabbits with a non-occlusive jugular vein thrombus composed of whole human plasma were :These results indicate that, after 1 to 3 half-lives of the IgG or Fab fragments, using combinations of 2 or 3 monoclonal antibodies, thrombus to blood ratios of isotope of 5 to 7 are obtained. Such signal/noise ratios are sufficient for in vivo detection by exteijil gamma scintigraphy. This was preliminarily confirmed using 123I-labeled Fab fragments of the three antibodies in rabbits.

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In Vivo Penetration of Experimentally Produced Clots by Monoclonal Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613937 ◽

2000 ◽

Vol 83 (06) ◽

pp. 882-886 ◽

Cited By ~ 1

Author(s):

T. A. Edgell ◽

P. M. Webbon ◽

P. J. Gaffney ◽

F. J. McEvoy

Keyword(s):

Monoclonal Antibodies ◽

Jugular Vein ◽

Human Fibrinogen ◽

Contralateral Limb ◽

Thrombotic Disease ◽

Gain Access ◽

Circulating Antibody

SummaryAntifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of two antifibrin mabs (NIB 1H10 and NIB 12B3) to penetrate occlusive clots in vivo was investigated Both mabs react with human fibrin but not with human fibrinogen nor with the fibrin or fibrinogen from the species used in this study.Two heterologous animal (sheep and rabbit) thrombus models were used. Clots in both cases were made within isolated vein segments using a mixture of human and native fibrinogen. The clots in sheep veins were observed radiographically and found to be occlusive for a mean of 4.2 ± 2.2 days and thereafter appeared only partially occlusive. When targeted in their occlusive phase 131I labelled mab accumulated in the clot reaching a maximum ratio of 1.82 ± 0.42 when compared to counts in homologous sheep clots in the contralateral limb. It was confirmed in the rabbit jugular vein model that total occlusivity did not prevent antibody accumulation in the heterologous clot by injecting the fibrin specific mab IH10 and examining the clot excised after 1, 6 and 24 h using immunofluorescence. In a further series of similar experiments 125I labelled mab 1H10 was used and detected using autoradiography. Both sets of experiments indicated that penetration of occlusive clots by the antibody occurred and that considerable accumulation was present at 6 and 24 h.The results indicate that a circulating antibody can readily gain access to experimentally produced clots in occluded veins.

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MONOCLONAL ANTIBODIES OF THE IgM AND IgG CLASS SPECIFIC FOR CROSSLINKED FIBRIN DEGRADATION PRODUCTS

10.1055/s-0038-1643651 ◽

1987 ◽

Author(s):

P J Gaffney ◽

L J Creighton ◽

A Curry ◽

B MacMahon ◽

R Thorpe

Keyword(s):

Monoclonal Antibodies ◽

Thrombolytic Therapy ◽

Epitope Mapping ◽

Degradation Products ◽

Subcutaneous Route ◽

Fibrin Degradation Products ◽

Conformational Epitopes ◽

Immunoglobulin Class Switching ◽

Unique Specificity ◽

D Dimer

Monoclonal antibodies (mabs) to crosslinked fibrin degradation products (XL-FDP) having the general formula D/Y[X]nY/D (known as X-oligomer) and D-D (known as D dimer) have been raised in balb/C mice by both a novel mtrasplenic and a conventional subcutaneous route of immunisation and by combinations of both these procedures. Mabs to X-oligomers (NIBn 52 and NIBn 123) obtained by an intrasplenic procedure have been demonstrated to crossreact only with X-oligomer in a 2-site ELISA procedure and not with D dimer or whole fibrinogen and have been shown to be of value m the examination of clinical material obtained from patients with various types of thrombosis and have also been useful in monitoring the efficacy of thrombolytic therapy. The X-oligomer mabs are immunoglobulins of the M class. It was demonstrated that their unique specificity for conformational epitopes on the large X-oligomer fragments does not reside in the IgM structure since alterative immunisation procedures have been used to generate mabs of the IgG class which have the same specificity. Using immunoglobulin class switching in culture rather than during immunisation was suggested by certain cell lines which produced both IgM and IgG specific for X-oligomer. This latter point needs rigorous validation.Immunoglobulin G type mabs to highly purified D dimer were raised by conventional subcutaneous immunisation of balb/C mice. One of these, NIBn-11, was found to crossreact with PVC-immobilised X-oligomer and D dimer but not with fibrinogen. However NIBn-11 did not bind to D dimer in a 2-site ELISA procedure while crossreactmg quite avidly with X-oligomer. This suggests that the D dimer epitope to which NIBn-11 is directed is expressed in some conformations and not m others and that these conformations are always expressed in the complex X-oligomer group of fragments. These mabs, whilst of value in measuring certain unique fibrin fragments m plasma, are useful in the epitope mapping of fibrinogen/fibrin and their plasmm-mediated

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The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

Frontiers in Plant Science ◽

10.3389/fpls.2021.671728 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pia Gattinger ◽

Shiva Izadi ◽

Clemens Grünwald-Gruber ◽

Somanath Kallolimath ◽

Alexandra Castilho

Keyword(s):

Monoclonal Antibodies ◽

Fusion Proteins ◽

Degradation Products ◽

Therapeutic Proteins ◽

Cost Effective ◽

Full Length ◽

Peptide Sequence ◽

Reporter Protein ◽

The Stability

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

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Factors Influencing The Structure Of Terminal Plasmin Degradation Products Of Human Fibrinogen And Fibrin

10.1055/s-0038-1653181 ◽

1981 ◽

Author(s):

W Nieuwenhuizen ◽

A Vermond ◽

F Haverkate

Keyword(s):

Protective Effect ◽

Calcium Ions ◽

Degradation Products ◽

Iminodiacetic Acid ◽

Human Fibrinogen ◽

Factors Influencing ◽

Separate Effect ◽

The Media ◽

Carboxy Terminal ◽

D Dimer

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 plasmin attack is rather independent of the composition of the media (e.g., also observed in 2 M urea and/or high plasmin activities).Several compounds such as EDTA, EGTA, citrate and iminodiacetic acid appear to have a separate effect, which is best observed at low plasmin concentrations and in the absence of Ca2+ . Under these conditions, 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.

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DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES

10.1055/s-0038-1643865 ◽

1987 ◽

Author(s):

J Abbink ◽

J Nuijens ◽

C Huijbregts ◽

E Hack

Keyword(s):

Monoclonal Antibodies ◽

Longitudinal Studies ◽

Human Plasma ◽

Dextran Sulfate ◽

Optimal Conditions ◽

Α2 Macroglobulin ◽

In Vitro Activation

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.

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Identification of D-Dimer-E Complex in Disseminated Intravascular Coagulation (DIC)

10.1055/s-0039-1684840 ◽

1979 ◽

Author(s):

A.N. Whitaker ◽

E.A. Rowe ◽

P.P. Masci ◽

P.J. Gaffney

Keyword(s):

Gel Electrophoresis ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Stable Complex ◽

Fibrin Degradation Products ◽

Pneumococcal Sepsis ◽

D Antigen ◽

D Dimer

D-dimer (D2), a product of the plasmin lysis of cross-linked (XL) fibrin, but not of non-XL fibrin or fibrinogen, has been identified in the plasma of patients with DIC due to amniotic fluid embolism. In vitro, D is involved with fragment E as a stable complex (D2-E) but D2 -E has not been identified in vivo before. Fibrin degradation products (FDP) were studied in a patient having fulminant postsplenectomy pneumococcal sepsis and DIC, by immunoprecipitation with anti-fibrinogen (f) and anti-fragment E and characterization by SDS polyacrylamide gel electrophoresis (PAGE). With both antisera soluble HMW fibrin complexes, D2 and E but no X, Y or D were obtained from serum. D2 and E were identified in the supernatant after removing partially XL HMW complexes and fibrinogen from plasma with 2.5 M β-alanine. The presence D antigen in the D2-E complex precipitated by monospecific anti-E was confirmed by crossed Ag-Ab electrophoresis. Crossed Ag-Ab electrophoresis of serum in agarose gave E peaks of slow mobility and no fast-moving free E was found. Thus, D2-E complex exists in vivo and its easy identification, proving the lysis of XL fibrin, would be of value in studying thrombosis. D2-E complex has been identified in other patients with sepsis but at lower concentrations than described above.

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The Immunosuppressive Activity of Plasmic Degradation Products of Human Fibrinogen

10.1055/s-0039-1682781 ◽

1977 ◽

Author(s):

T.S. Edgington ◽

L.K. Curtiss ◽

E.F. Plow

Keyword(s):

Cell Response ◽

Degradation Products ◽

Thymidine Uptake ◽

Immunosuppressive Activity ◽

Human Fibrinogen ◽

Peptide Fraction ◽

Intact Molecule ◽

Stimulation Of

Plasmic cleavage of human fibrinogen leads to generation of immunosuppressive activity not expressed by the intact molecule, and which is demonstrable in vitro and in vivo. This activity is not associated with the high molecular weight derivatives X, Y, D and E, but is present in the small dialyzable peptide fraction obtained from plasmic digestion. The peptides inhibit in a non-toxic fashion, the stimulation of 3H-thymidine uptake and blastogenesis of lymphocytes by phytohemagglutinin (PHA) and allogeneic cells (MLC) under conditions of both macrophage dependence and macrophage independence. The peptides also suppress the plaque-forming cell response of mice to sheep red blood cells in vivo. Approximately 30 μg peptides/culture leads to a 50% inhibition of the PHA and MLC systems, and approximately 400 μg/mouse produces a 50% suppression of the plaque-forming cell response. Intact fibrinogen chains exhibit negligible activity, but plasmic digests of Aα chain are suppressive. Consistent with derivation from the Aa chain was the demonstration that the activity was generated from limited plasmic digest of fibrinogen which produced fragment X, and this activity was soluble at 80°C for 10 minutes. The release of the active peptide by limited plasmic degradation, and the activity of these peptides at physiologic concentrations suggests that this system may be of importance in vivo in association with local fibrinogenolysis or fibrinolysis at sites of thrombosis. This has been in part substantiated by the experimental initiation of fibrinogenolysis in vivo with streptokinase.

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Plasma levels of D-dimer and fibrin degradation products correlate with bullous pemphigoid severity: a cross-sectional study

Scientific Reports ◽

10.1038/s41598-021-97202-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sijia Wang ◽

Mei Lu ◽

Zijun Zhao ◽

Xueting Peng ◽

Liang Li ◽

...

Keyword(s):

Bullous Pemphigoid ◽

Degradation Products ◽

Eosinophil Cationic Protein ◽

Control Group ◽

Cationic Protein ◽

Glucocorticoid Treatment ◽

Cross Sectional ◽

Fibrin Degradation Products ◽

Eosinophil Counts ◽

D Dimer

AbstractBullous pemphigoid (BP), the most frequent blistering dermatosis in the elderly, is associated with increased mortality. The severity of BP can be assessed by detecting the anti-BP180 immunoglobulin G (IgG) concentration, but the lab test is not available in many community clinics. BP patients are usually in a hypercoagulable state with increased levels of D-dimer and fibrin degradation products (FDPs). We aimed to evaluate the use of D-dimer and FDPs in assessing BP severity. We compared the levels of plasma D-dimer, plasma FDPs, eosinophil counts, eosinophil cationic protein, and serum anti-BP180 IgG concentration between 48 typical BP patients and 33 Herpes zoster (HZ) patients (control group). Correlational analyses were conducted to determine the relationships between the lab values and common BP severity markers. The plasma D-dimer and FDP levels were higher in BP patients than in HZ controls (D-dimer: 3297 ± 2517 µg/L vs. 569.70 ± 412.40 µg/L; FDP: 9.74 ± 5.88 mg/L vs. 2.02 ± 1.69 mg/L, respectively, P < 0.0001). Significant positive correlations were found between D-dimer/FDP levels and BP severity markers (i.e. anti-BP180 IgG concentration [D-dimer: r = 0.3928, P = 0.0058; FDP: r = 0.4379, P = 0.0019] and eosinophil counts [D-dimer: r = 0.3625, P = 0.0013; FDP: r = 0.2880, P = 0.0472]) in BP patients. We also found an association between FDP and urticaria/erythema lesions (r = 0.3016, P = 0.0372), but no other BPDAI components. In 19 BP patients with complete remission after systemic glucocorticoid treatment, D-dimer and FDP levels decreased post-therapy (D-dimer: 5559 ± 7492 µg/L vs. 1738 ± 1478 µg/L; P < 0.0001; FDP: 11.20 ± 5.88 mg/L vs. 5.13 ± 3.44 mg/L; P = 0.0003), whereas they did not in BP patients with treatment resistant. Plasma D-dimer and FDP are convenient markers to evaluate BP severity assistant on BPDAI and eosinophil counts. FDP is also helpful for inflammatory lesions in BP patients.

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