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.

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.

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.

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 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.

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

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.

D-Phe-Pro-Arg-Chloromethylketone: Its Potential Use in Inhibiting the Formation of In Vitro Artifacts in Blood Collected During Tissue-Type Plasminogen Activator Thrombolytic Therapy

1986 ◽  
Vol 56 (02) ◽  
pp. 160-164 ◽  
Author(s):  
M A Mohler ◽  
C J Refino ◽  
S A Chen ◽  
A B Chen ◽  
A J Hotchkiss
Keyword(s):  
Plasminogen Activator ◽  
Degradation Products ◽  
Significant Loss ◽  
Tissue Type ◽  
Blood Samples ◽  
Tissue Type Plasminogen Activator ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Potential Use

Summary In vitro artifacts due to proteolysis may occur in blood samples containing recombinant tissue-type plasminogen activator (rt-PA) due to continued activation of plasminogen to plasmin by rt-PA. The aim of this study was to identify a rapid inhibitor of rt-PA that would not interfere in assays designed to monitor thrombolytic events.When rt-PA was added at 5 μg/ml to whole blood and incubated at 25° C, fibrinogen decreased 50 percent, plasminogen levels decreased 90 percent and α2-antiplasmin decreased below detectable levels. If D-Phe-Pro-Arg-chloromethylketone (PPACK) or aprotinin were added before the addition of rt-PA there was no significant loss of fibrinogen. Only PPACK completely inhibited changes in fibrin degradation products, plasminogen and α2-antiplasmin. PPACK was also found to inhibit the binding of rt-PA to plasma protease inhibitors in vitro.Rhesus monkeys were infused with rt-PA and blood samples were taken with either PPACK or aprotinin in the collection syringe. There was a significant increase in the recovery of immunoreactive rt-PA and consistent measures of fibrinogen, FDPs, plasminogen, and α2-antiplasmin in the PPACK group as compared to the aprotinin group which indicates that PPACK will prevent the in vitro formation of artifacts due to the presence of active rt-PA

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.

Monoclonal Antibodies to Tissue-Type Plasminogen Activator which Prolong its Clearance In Vivo

1989 ◽  
Vol 61 (02) ◽  
pp. 259-261
Author(s):  
Thomas M Reilly ◽  
Robert M Knabb ◽  
Andrew T Chiu ◽  
David L Bradfute ◽  
Pieter B M W M Timmermans
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Half Life ◽  
Cell Receptor ◽  
Tissue Type ◽  
Human Hepatoma Cells ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryThree murine monoclonal antibodies to tissue-type plasminogen activator (t-PA) were evaluated for their effects on the binding of iodinated t-PA to cultured human hepatoma cells (Hep G2), and on extending the half-life of t-PA injected into rabbits. Two of the antibodies, AE5 and EG2, significantly inhibited t-PA binding in vitro, and extended the in vivo half-life of t-PA four to five-fold. A third antibody, BA10, which had a much smaller inhibitory effect on t-PA binding, had no influence in extending t-PA’s half-life. MOPC-21, a control antibody not directed to t-PA, had no effect on either test. Our results are the first to correlate different compounds’ effects on t-PA binding with their ability to retard t-PA clearance in vivo, and provide additional evidence for the importance of a liver cell receptor in the t-PA clearance process.

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.

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