scholarly journals Production and characterization of a monoclonal antibody reactive with a specific neoantigenic determinant (comprising B beta 54-118) in degradation products of fibrin and of fibrinogen

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 437-441
Author(s):  
PW Koppert ◽  
J Koopman ◽  
F Haverkate ◽  
W Nieuwenhuizen
Keyword(s):  
Degradation Products ◽  
Blood Clot ◽  
Immunoaffinity Chromatography ◽  
Tissue Type ◽  
Spleen Cells ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Complete Lysis ◽  
Fibrin Monomers

Balb/c mice were immunized with a mixture of fibrin degradation products (XDPs) prepared by complete lysis of a human blood clot by tissue-type plasminogen activator and purified by immunoaffinity chromatography. Spleen cells of the mice were fused with P3 X 63 Ag 8653 myeloma cells. A clone (FDP 14) was selected that produces monoclonal antibodies (MoAbs) of the IgG1 kappa type that react with a neoantigenic determinant exposed in these XDPs, but not in intact fibrinogen or in fibrin monomers. Furthermore, the MoAb is reactive with some pure, individual degradation products of fibrinogen (fragments X, Y, E, and the N-terminal disulphide knot) and with the fibrinogen B beta-chain but not with A alpha- and gamma-chains or with fragments D, FCB-2 and FCB-3. Comparison of the known primary structures of these fibrinogen fragments indicates that the stretch B beta 54–118 comprises at least an important part of the epitope recognized by FDP-14. Apparently, this stretch contributes importantly to a neoantigenic determinant that is not functional in intact fibrinogen and fibrin monomer and that can be made functional by reduction of fibrinogen, or by digestion with plasmin or CNBr.

scholarly journals Production and characterization of a monoclonal antibody reactive with a specific neoantigenic determinant (comprising B beta 54-118) in degradation products of fibrin and of fibrinogen

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 437-441 ◽  
Author(s):  
PW Koppert ◽  
J Koopman ◽  
F Haverkate ◽  
W Nieuwenhuizen
Keyword(s):  
Degradation Products ◽  
Blood Clot ◽  
Immunoaffinity Chromatography ◽  
Tissue Type ◽  
Spleen Cells ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Complete Lysis ◽  
Fibrin Monomers

Abstract Balb/c mice were immunized with a mixture of fibrin degradation products (XDPs) prepared by complete lysis of a human blood clot by tissue-type plasminogen activator and purified by immunoaffinity chromatography. Spleen cells of the mice were fused with P3 X 63 Ag 8653 myeloma cells. A clone (FDP 14) was selected that produces monoclonal antibodies (MoAbs) of the IgG1 kappa type that react with a neoantigenic determinant exposed in these XDPs, but not in intact fibrinogen or in fibrin monomers. Furthermore, the MoAb is reactive with some pure, individual degradation products of fibrinogen (fragments X, Y, E, and the N-terminal disulphide knot) and with the fibrinogen B beta-chain but not with A alpha- and gamma-chains or with fragments D, FCB-2 and FCB-3. Comparison of the known primary structures of these fibrinogen fragments indicates that the stretch B beta 54–118 comprises at least an important part of the epitope recognized by FDP-14. Apparently, this stretch contributes importantly to a neoantigenic determinant that is not functional in intact fibrinogen and fibrin monomer and that can be made functional by reduction of fibrinogen, or by digestion with plasmin or CNBr.

A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

1988 ◽  
Vol 59 (02) ◽  
pp. 310-315 ◽  
Author(s):  
P W Koppert ◽  
E Hoegee-de Nobel ◽  
W Nieuwenhuizen
Keyword(s):  
Enzyme Immunoassay ◽  
Degradation Products ◽  
Blood Clot ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Strong Positive Reaction ◽  
Sandwich Type ◽  
Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

A NEW QUANTITATIVE ENZYME-IMMUNOASSAY FOR FIBRIN DEGRADATION PRODUCTS (FbDP) IN PLASMA, BASED ON MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
P W Koppert ◽  
E Hoegee-de Nobel ◽  
W Nieuwenhuizen
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Degradation Products ◽  
Blood Clot ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Strong Positive Reaction ◽  
Sandwich Type

We have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes. The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the catching antibody, binds both fibrinogen degradation products (FbgDP) and FbDP. It has its epitope in the E-domain of the fibrinogen molecule on the BB-chain between amino acids 54-118 (Blood 68, 437, 1986). Antibody DD-13 was raised using D-dimer as antigen and was used as a tagging antibody, conjugated with horse-radish peroxidase.A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard.The EIA does not detect FbgDP i.e. purified fragments X, Y, D:E complexes or FbgDP in plasma treated in vitro with streptokinase. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseases. In combination with the assays previously developed by us for FbgDP (Thromb. Haemostas. 1987, in press) and for the total amount (TDP) of FbgDP + FbDP in plasma (J. Lab. Clin. Med. 1987, in press), we are now able to study the composition of TDP in terms of FbgDP and FbDP in patients.

PLASMA LEVELS OF FRAGMENT D-DIMER OF CROSSLINKED FIBRIN DURING THROMBOLYTIC THERAPY WITH RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR

1987 ◽  
Author(s):  
P Declerck ◽  
P Mombaerts ◽  
P Holvoet ◽  
D Collen
Keyword(s):  
Thrombolytic Therapy ◽  
Plasma Levels ◽  
Degradation Products ◽  
Fibrin Clot ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Rate Of Increase ◽  
Significant Difference ◽  
D Dimer

Plasma levels of crosslinked fibrin degradation products (XLDP) were measured before and at the end of the administration of rt-PA (40 to 100 mg over 1.5 to 8 hours) in healthy volunteers (n=5) and patients with deep venous thrombosis (DVT) (n=8), pulmonary embolism (PE) (n=16)and myocardial infarction(MI)(n=10). Determinations were performed using our newly developed ELISA, specific for crosslinked fibrin derivatives, based on two monoclonal antibodies (15C5 and 8D3H2) raised against purified human fragment D-dimer. All plasma samples were collected on citrate and trasylol. Results are expressed as mean and range of D-dimer equivalents (μg/ml).Baseline levels in patients with MI are only slightly elevated. The increased levels inDVT and PE are in agreement with previous studies. After infusion of rt-PA a small increase of XLDP is seen even innormal subjects. A very marked increasof XLDP is detected in patients with PE and DVT but not in patients with MI. This may reflect differences in the amounts of fibrin clot dissolved in these patient groups.No significant correlation was found between the increase of XLDP and success of therapy, although a significant difference in D-dimer levels was formed between the two groups with PE: successful (n=ll): 116 (range 61-192) vs. unsuccessful (n=5): 68 (36-155).Thus, XLDP are already elevated under baseline conditions in patients with DVT and PE and increase very markedly during thrombolytic therapy. The absolute levels after thrombolytic therapy do not strictly correlate with success of therapy. It could be useful to measure D-dimer levels during early stages of therapy, because the rate of increase of XLDP levels might correlate with the efficacy of thrombolytic treatment.

Sustained Fibrinolysis After Administration of t-PA Despite Its Short Half-Life in the Circulation

1987 ◽  
Vol 57 (01) ◽  
pp. 035-040 ◽  
Author(s):  
Paul R Eisenberg ◽  
Laurence A Sherman ◽  
Alan J Tiefenbrunn ◽  
Philip A Ludbrook ◽  
Burton E Sobel ◽  
...  
Keyword(s):  
Half Life ◽  
Fibrinolytic Activity ◽  
Degradation Products ◽  
Clinical Success ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Fibrin Degradation Products ◽  
Short Half Life ◽  
Type Plasminogen Activator ◽  
The Difference

SummaryTo characterize the duration of the fibrinolytic response to tissue-type plasminogen activator (t-PA) and streptokinase (SK) in patients with acute myocardial infarction we serially assayed crosslinked fibrin degradation products (XL-FDP) and Bβ15-42 fibrinopeptide. Use of specific monoclonal antibodies permitted quantification and differentiation of fibrin from fibrinogen degradation products. Marked elevations of XL-FDP occurred within 1 hour after administration of t-PA (n = 13) or SK (n = 35) to >1000 ng/ml in 79% of the patients. All patients given t-PA exhibited elevations of XL-FDP >1000 ng/ml, most exhibited values >5000 ng/ml (79% of patients). In contrast 6 of the patients given SK failed to exhibit XL-FDP >1000 ng/ml. XL-FDP >5000 ng/ml occurred in only 14%. The difference in the response to t-PA compared to SK was particularly striking 7 hours or more after administration of activator at which time XL-FDP were markedly elevated in patients given t-PA (5821 ± 1683 ng/ ml) compared with decreasing values in patients given SK (2924 ± 1186 ng/ml) (p <0.01). Levels of Bβ315-42 were significantly higher after t-PA compared with SK beginning 3 hours after treatment, consistent with a greater intensity of fibrinolytic response to t-PA. Marked elevations of this short lived degradation product of fibrin (t1/2 = 10-20 minutes) in the samples drawn late after administration of t-PA (44.3 ±12.8 nM) but not after SK (11.7 ± 4.5 nM) confirmed prolonged fibrinolytic activity of plasmin after t-PA. There was no discernible relationship between the extent of fibrinolysis as assessed by XL-FDP and Bβ 15-42 and the total dose of t-PA administered or the duration of the infusion. Elevations of XL-FDP invariably occurred after SK, and were not significantly different in patients with or without recanalization. Thus “clinical success” of coronary thrombolysis appears to depend on a favorable balance between thrombosis and fibrinolysis rather than the intensity of fibrinolysis alone. The prolonged fibrinolytic activity after t-PA appears to reflect the enhanced binding of this activator to fibrin and is likely to result in more sustained and hence more effective fibrinolysis with t-PA compared to SK despite the short half-life of t-PA (t1/2 = 6 minutes) in the circulation.

scholarly journals The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1189-1192 ◽  
Author(s):  
NJ de Fouw ◽  
F Haverkate ◽  
RM Bertina ◽  
J Koopman ◽  
A van Wijngaarden ◽  
...  
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Degradation Products ◽  
Blood Clot ◽  
Activated Protein C ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Fibrin Degradation Products

Abstract The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.

DDE COMPLEX DETERMINATION AS A SPECIFIC MARKER FOR FIBRINOLYSIS

1987 ◽  
Author(s):  
J Soria ◽  
C Soria ◽  
Me Mirshahi ◽  
S Mirshahi ◽  
M Mirshahi ◽  
...  
Keyword(s):  
Degradation Products ◽  
Clinical Investigation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Specific Marker ◽  
Fibrin Degradation Products ◽  
Pathological Conditions ◽  
Fibrin Monomer ◽  
Type Plasminogen Activator ◽  
Conventional Elisa

Monoclonal antibodies (McAb) reacting with fibrin degradation products (FbDP), but not with fibrinogen have been produced in order to determine specifically FbDP directly in plasma. Most of the McAb available however do also react with fragment D. Our anti D neo McAb reacts about 10 times less with fragment D than with FbDP but does not react with fibrinogen, fragment X or Y.In clinical investigation, even in pathological conditions in which there is a great release of tissue-type plasminogen activator (tpA), we have shown that fragment D is not generated in patients plasma. Therefore, the reactivity of our McAb with fragment D did not alter the specificity of FbDP assay.On the contrary, using polyacrylamide gel electrophoresis in the presence of SDS followed by immunorevelation, we have evidenced that fragment D is generated in patients undergoing thrombolytic therapy even with rtpA. Therefore, using conventional Elisa procedure (capture of FbDP on polystyrene-immobilized anti D neo antibody and detection by peroxidase-labelled anti fragment D immunoglobulins), the presence of fragment D in patients plasma leads to an overevaluation of FbDP. To avoid this overestimation we have modified the Elisa procedure. The structure of FbDP was taken into acount in order to render the technique specific of FbDP. In FbDP fragment D coming from one fibrin monomer is always associated with fragment E from another fibrin monomer, as DDE complex for example. Therefore after capture of fragment D by the polystyrene-bound anti D neo McAb, FbDP were specifically revealed by peroxidase-labelled anti E antibody (polyclonal or monoclonal anti E may be used). For this reason, this test was named DDE determination and DDE determination can be used in any circumstances to evaluate fibrin degradation.

DISSOCIATION BETWEEN DDE LEVEL AND RECANALIZATION IN PATIENTS UNDERGOING THROMBOLYTIC THERAPY FOR MYOCARDIAL INFARCTION. A POSSIBLE EXPLANATION

1987 ◽  
Author(s):  
Mc Mirshahi ◽  
J Soria ◽  
C Soria ◽  
Ma Xi ◽  
M Mirshahi ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Monoclonal Antibody ◽  
Thrombolytic Therapy ◽  
Degradation Products ◽  
Careful Analysis ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Almost All

The aim of this work was to determine whether plasma fibrin degradation products (FbDP) level may be used as a marker of recanalization in patients undergoing thrombolytic therapy for myocardial infarction. Samples were collected in patients treated by a 90 minutes infusion of 60 mg recombinant tissue type plasminogen activator (rtpA). Plasma FbDP (DDE complexes) were specifically and reliably determined by Elisa using a personal monoclonal antibody. Additional identification of FbDP and fibrinogen derived fragment D was performed by SDS PAGE followed by their detection using an anti D neo monoclonal antibody.Fragment D was only evidenced up to the end of rtpA infusion. Concerning FbDP levels : prior to treatment, each patient presented FbDP levels within the normal range. After rtpA infusion, FbDP were increased in almost all cases, but our results clearly demonstrated that this increase was not correlated with coronary recanalization : on one hand, FbDP (D dimer and YD complex) were increased at the end of rtpA infusion in patients for whom the treatment was efficient as proved by angiography but also in five of seven patients who did not reperfuse. On the other hand, in most cases even in patients presenting a good recanalization at the end of rtpA infusion, a further increase in FbDP was noted even 6 hours after the end of the infusion.It is therefore suggested that FbDP came either from a degradation of fibrin from different sources that coronary thrombus or coagulation in coranary artery was going on. A careful analysis of a possible activation of coagulation during thrombolytic therapy is under investigation.In this way in patients under thrombolytic therapy, we propose to investigate the balance between coagulation and fibrinolysis by determining at the same time the level of plasma fibrinopeptide A and DDE complexes.

Fibrin Metabolism in Patients with Acute Myocardial Infarction During and After Treatment with Tissue-Type Plasminogen Activator

1988 ◽  
Vol 60 (03) ◽  
pp. 428-433 ◽  
Author(s):  
Michael E Ring ◽  
Samuel M Butman ◽  
Denise C Bruck ◽  
William M Feinberg ◽  
James J Corrigan
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Molecular Markers ◽  
Plasminogen Activator ◽  
Degradation Products ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator

SummaryIn order to define some of the determinants of successful thrombolysis and reocclusion during fibrinolytic therapy for acute myocardial infarction (AMI), specific molecular markers of fibrin metabolism were serially measured in 15 patients with AMI treated with tissue-type plasminogen activator (t-PA). Fibrin formation was assessed by measurement of fibrinopeptide A (FpA) and fibrinolysis by assay of B-P peptides 1—42 and 15—42 and crosslinked fibrin degradation products (XDP). At baseline, FpA levels were high while markers of fibrinolysis were near normal. Following a 90-minute infusion of t-PA (0.5—1.1 mg kg−1 hr−1), all markers of fibrinolysis increased. Levels of FpA remained elevated despite heparin at the initiation of cardiac catheterization. None of these markers discriminated between patients with successful reperfusion from those without. At 4 hours, B-β 15—42 peptide and XDP levels remained elevated suggesting persistence of fibrinolysis beyond the short circulatory half-life of t-PA. FpA levels at 4 hours were lower in patients who underwent acute coronary angioplasty compared to those who received additional low dose t-PA (12.3 ± 4.5 vs. 30.4 ± 5.5 ng/ ml, p <0.05). By 48 hours, markers of fibrinolysis had returned toward normal except in 2 patients with persistently elevated B-P 15—42 peptide levels who suffered reocclusion on days 5 and 6 (75 and 44 vs. 29 ± 3 nM, p <0.005). In conclusion, molecular markers of fibrin metabolism during fibrinolytic therapy may provide clinically relevant data.

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