Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA.

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

Development of A Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers.

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.

Age-Adjusted D-Dimer in the Prediction of Pulmonary Embolism: Does a Normal Age-Adjusted D-Dimer Rule Out PE?

Risk assessment for pulmonary embolism (PE) currently relies on physician judgment, clinical decision rules (CDR), and D-dimer testing. There is still controversy regarding the role of D-dimer testing in low or intermediate risk patients. The objective of the study was to define the role of clinical decision rules and D-dimer testing in patients suspected of having a PE. Records of 894 patients referred for computed tomography pulmonary angiography (CTPA) at a University medical center were analyzed. The clinical decision rules overall had an ROC of approximately 0.70, while signs of DVT had the highest ROC (0.80). A low probability CDR coupled with a negative age-adjusted D-dimer largely excluded PE. The negative predictive value (NPV) of an intermediate CDR was 86-89%, while the addition of a negative D-dimer resulted in NPVs of 94%. Thus, in patients suspected of having a PE, a low or intermediate CDR does not exclude PE; however, in patients with an intermediate CDR, a normal age-adjusted D-dimer increases the NPV.

Effect of fibrin degradation products on fibrinolytic process.

Fibrin clot lysis by plasminogen/plasmin system results in fibrin degradation products formation with subsequent release into bloodstream. The fragments contain specific binding sites for fibrinolytic system components and can interact with them. In this study, we investigated the way in which fibrin fragments effect fibrinolytic process. We have shown that high molecular weight products of fibrin degradation and fibrin fragments of DDE-complex and DD, but not end product Е3, stimulate plasmin formation. Additionally, components of DDE-complex mixture of fragments Е1 and Е2 have potentiation ability. The intermediate fibrin fragments hmFDPs and DDE attenuate clot lysis by plasmin and hmFDPs protect plasmin from α2-antiplasmin inhibition but under further fragmentation to endpoint fibrin fragments loose this ability. The plasma inhibitors reduce fibrinolytic system activity generated by the degradation products. Thus, fibrin fragments formed during the clot lysis can bind and move out fibrinolytic system components from clot volume and in this way result in clot resistance to hydrolysis.

Pulmonary embolism risk assessment screening tools: the interrater reliability of their criteria.

BACKGROUND: Diagnostic evaluation for suspected pulmonary embolism (PE) is challenging. Dimerized plasmin fragment D (D-dimer) assays are increasingly used but have been validated only in "low-risk" patients. The accurate interpretation and application of risk assessment criteria are critical to the appropriate use of D-dimer. We sought to determine the interrater agreement of attending and third-year resident emergency medicine physicians in the specific elements of the Canadian and the Charlotte risk stratification tools and their clinical application. METHODS: We prospectively enrolled a convenience sample of patients presenting to an urban university emergency department with suspected PE. Standardized data collection sheets were used by an attending physician and a third-year resident physician to determine the presence or absence of risk factors included in published PE prediction instruments. Each physician was blinded to the other's results and the patients' D-dimer result. Interrater agreement was measured using kappa statistics (with 95% confidence intervals). RESULTS: Two hundred seventy-one patients were screened. The kappa scores for each risk criterion were as follows: previous deep vein thrombosis, 0.90 (95% confidence interval, 0.83-0.97); malignancy, 0.87 (0.76-0.97); deep vein thrombosis symptoms, 0.54 (0.39-0.70); immobilization, 0.41 (0.26-0.57); unexplained hypoxia, 0.58 (0.42-0.74); tachycardia, 0.94 (0.89-0.98); hemoptysis, 0.76 (0.51-1.0); and PE more likely than another diagnosis, 0.50 (0.36-0.64). CONCLUSIONS: Interrater agreement was only fair for several important risk criteria. Small differences in determining pretest probability can lead to significant variability in risk assessment and how, or whether, the diagnosis of PE is evaluated. This study raises questions about the reliability and applicability of published PE screening criteria in clinical settings.

Detection of anticoagulant activities of isolated canine fibrinogen degradation products X, Y, D and E using resonance thrombography.

The objective of the study was to examine the influence of fibrinogen degradation products (FDPs) on the resonance thrombogram (RTG). Different concentrations (up to 5 g/l plasma) of purified canine FDP X, FDP Y, FDP D and FDP E were added to the blood of healthy dogs and the RTG was measured. Of the examined RTG parameters [reaction time, fibrin formation time (RTG-f), and fibrin amplitude], FDP X, FDP Y, and FDP D showed the clearest effects on RTG-f. RTG-f was on the average prolonged by addition of 0.06 g/l FDP Y, 0.15 g/l FDP X, and 0.17 g/l FDP D to the upper limit of the reference range. Based on the gram amounts of the added FDP, fragment Y also showed in other RTG parameters the most marked effect, followed by FDP X and FDP D. FDP E showed no substantial effect rS [Spearmans rank correlation coefficient (< 0.3, P > or = 0.5)] in any of the examined RTG parameters. The closest correlation between FDP concentration and the result of the different RTG parameters was found for the RTG-f (FDP X, rS = 0.819; FDP Y, rS = 0.795; FDP D, rS = 0.764). The results indicate that RTG is a useful screening test for the detection of increased FDP concentrations.

Isolation of bound thrombin consisting of thrombin and fibrin N-terminal fragment from clot lysate.

It has been reported that thrombin is liberated from fibrin clots by the action of fibrinolytic enzymes. It has also been reported that the liberated thrombin complexes with fibrin fragment E or (DD)E, which are denoted as bound thrombin. However, bound thrombin has not been isolated from clot lysate, and the structural characteristics of isolated bound thrombin have not been specified. In this study, we attempted to isolate the bound thrombin from clot lysate and to clarify its structural features. Rabbit fibrinogen was clotted with bovine thrombin, and clot lysate was prepared with urokinase. The bound thrombin was isolated from clot lysate by serial chromatography using a Sepharose 4B column immobilizing an anti-bovine thrombin antibody and a Sepharose 4B column immobilizing an anti-rabbit fibrinogen antibody. SDS-PAGE under unreduced conditions demonstrated that there were two different protein bands in the isolated bound thrombin. On a C4 reverse-phase HPLC, the bound thrombin from clot lysate was resolved by 4 M urea into alpha-thrombin and a fibrin fragment, the N-terminal regions of which were identified as alpha-, beta- and gamma-chains. Thus, in the bound thrombin, thrombin molecule would bind to rabbit fibrin fragment consisting of N-terminal central domain.

Fibrinogen is a component of a novel lipoprotein particle: factor H-related protein (FHRP)-associated lipoprotein particle (FALP).

We have previously described a novel lipoprotein particle consisting of phospholipids, apolipoprotein A-I (apoAI), lipopolysaccharide binding protein (LBP) and Factor H-related proteins (FHRP), and we termed these particles FALP (FHRP-associated lipoprotein particles). Highly purified preparations of FALP contain variable amounts of an unidentified polypeptide triplet of Mr approximately 85,000 (tp85). Here we report that tp85 represents fragment D of fibrinogen, as confirmed by N-terminal amino acid sequencing and Western blot analysis with an antifibrinogen antibody. The physical association of fibrinogen with other components of FALP in plasma was further confirmed by sandwich ELISA by using monoclonal antibodies against apoAI, FHRP or LBP to capture the particles and polyclonal antifibrinogen as the detecting antibody. Furthermore, affinity chromatography with anti-FHRP-1-specific IgG showed that fibrinogen is co-immunodepleted with FALP and approximately 17% of total plasma fibrinogen are bound to FALP. LBP is a lipid transfer protein that moves lipopolysaccharide (LPS) to a binding site on CD14 or high-density lipoprotein (HDL). To determine whether fibrinogen affects the lipid transfer activity of LBP on FALP, this activity was measured in FALP prepared with and without fibrinogen. Neither activity of LBP was affected by fibrinogen. The abundance of FALP suggests, instead, an effect of FALP on the function or clearance of fibrinogen or fragment D. (Blood. 2000;95:198-204)

Facile purification of fibrinogen fragments using a computer-based model with general applicability to the generation of salt gradients.

We and others have recently shown that specific fragments of cross-linked fibrin affect cell behavior. In order to develop a facile method for the preparative scale purification of fibrin fragment D dimer, a simple gradient generating system for conventional chromatography was developed and validated, and methods of fibrin fragment D dimer purification were compared. The experimentally measured salt concentration/time relationship fell directly on the model-predicted line. Model-predicted changes in the reservoir volume and/or salt concentration in the limit buffer affected both the initial slope and the shape of the concentration/time relationship. This gradient generation method was used to separate the D domains of fibrin(ogen) from the amino terminal region E domain using anion-exchange chromatography. While the predicted salt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by Coomassie-stained SDS-PAGE and by immunoblotting. In conclusion, a facile, user-friendly, computer-based method to predict and generate salt gradients was written and validated by direct experimentation. While fibrinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pH-based separation technique.

Interaction of heparin with fibrinogen using surface plasmon resonance technology: investigation of heparin binding site on fibrinogen.

Heparin is widely used as an antithrombotic drug, having excellent anticoagulant properties. However in certain clinical situations heparin's efficacy seems to be somewhat limited. Despite the administration of heparin there is a high incidence of reocclusion of coronary arteries following thrombolytic therapy, and it has been observed that a significant number of patients receiving heparin treatment still exhibit thrombus extension. Although it is well established that the in vitro and in vivo anticoagulant activities of heparin is mediated via the potentiation of the major coagulation inhibitor, antithrombin III (ATIII), some in vivo antithrombotic mechanisms are not fully understood. There is poor correlation between the anticoagulant activity of heparin as measured by in vitro assays and their in vivo antithrombotic efficacy. This may be due to heparin being targeted to many blood constituents whose resultant activities on the coagulation system have not been measured as yet. The antithrombotic activity of heparin as well as the pathogenesis of bleeding complications during heparin treatment cannot be completely explained by the inhibition of blood coagulation factors. Platelet dysfunction and acceleration of fibrinolytic process have been implicated as additional factors involved. Recently a number of reports have suggested that the inhibition of the antithrombotic activity of heparin in these clinical situations may be due to the interaction of heparin with other plasma proteins specifically with fibrin(ogen) present in the thrombus. Despite the possible pathophysiological significance of heparin-fibrin(ogen) interaction, little is known about the physicochemical aspects of this reaction. In this study an attempt was made to locate where heparin binds to fibrin(ogen), using various isolated structural domains from the plasmin-mediated digests of fibrinogen and the individual chains of fibrinogen. The BIALITE system (Pharmacia Biosensor AB, Uppsala, Sweden) was employed for such a study. This utilises the Surface Plasmon Resonance (SPR) phenomenon and allows a direct quantitative analysis of the label-free molecular interaction, in real-time, from which association and dissociation rate constants can readily be obtained.

Variable immunoreactivity of D-dimer preparations for monoclonal antibody DD-3B6/22.

The monoclonal antibody DD-3B6/22, which is specific for crosslinked fibrin breakdown products, is used clinically in the diagnosis and monitoring of certain thrombotic conditions and has potential as a delivery agent for in vivo clot localisation. Laboratory preparations of D-dimer are used as reference standards in DD-3B6/22 assay systems. In this study, four D-dimer preparations, produced using a standardised method, were presented to the monoclonal antibody, DD-3B6/22 in different antigenic formats and immunoreactivity was assessed using a variety of methods. The results show a variability in reactivity of the D-dimer samples to DD-3B6/22; one preparation (QUT2) was immunoreactive only when immobilised on a surface and not when in solution. The implications of variable immunorecognition of target antigens are discussed with regard to diagnostic standards and in vivo clot localisation.

[Preparation and separation of milligram amounts of canine fibrin(ogen) degradation products (fdp) X, Y, D and E]. / Präparation und Reinigung von Milligramm-Mengen der caninen Fibrin(ogen)-Spaltprodukte (FSP) -X, -Y, -D und -E.

In the present investigation we first produced canine fibrinogen degradation products (FDP) following two optimized degradation protocols. These FDP-mixtures, which were alternatively enriched with X and Y fragments or D and E fragments, were purified further to individual FDP X, -Y, -D, and -E with > 95% purity by the means of two low pressure column chromatographic techniques (size exclusion chromatography and anionexchanger chromatography). With this techniques the FDP D could be separated into four different D subfractions. No satisfactory results were yielded by hydrophobic interaction chromatography (HIC) with C5-Alkylsuperose, chromato-focusing and separations with hydroxyapatit. The observed strong binding of fragment E on hydroxyapatit probably points to the maintenance of the calcium binding site on the prepared canine E-fragment.

Crystallization of fragment D from human fibrinogen.

Fragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide-linked chains (alpha' beta' gamma' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 A; the space group is P2(1), unit cell dimensions a = 108 A, b = 48 A, c = 167 A, beta = 106 degrees. Fragment D was also co-crystallized with the ligand GPRP-amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 A, b = 82 A, c = 432 A.

Deficient antithrombin III activity and enhanced fibrinolysis in patients with liver disease: evidence against a cause-effect relationship.

To investigate the pathogenesis of fibrinolysis in liver disease, antithrombin III (AT III) activity, prothrombin fragment (F1 + 2) and d-dimer (D-DI) were measured in 50 patients with liver disease and in 17 healthy controls. Moreover, 4 patients with cirrhosis were randomly assigned to receive either an intravenous infusion of AT III (at two different dosages) or placebo, with a crossover design. Increased levels of D-DI were detected in patients with cirrhosis and hepatocellular carcinoma in comparison both with control subjects and with patients with acute hepatitis or mild chronic liver disease. An inverse correlation was observed between AT III and D-DI (r = -0.755, P < 0.001, simple linear regression), while no correlation was found between D-DI or AT III and F1 + 2. The correlation of the deficiency of AT III activity by infusion of human AT III did not result in any significant change (P0.10, analysis of variance for repeated measures) of the plasma concentration of either D-DI or F1 + 2, in comparison to placebo. Thus, advanced forms of chronic liver disease, but not acute hepatitis and mild forms of chronic liver disease, are associated with increased plasma concentrations of markers of fibrinolysis, which are inversely correlated with AT III activity. However, the correction of the deficient AT III activity does not affect the plasma concentration of either D-DI or F1 + 2, thence not supporting the hypothesis that enhanced fibrinolysis in advanced liver disease is the result of low-grade disseminated intravascular coagulation.

The development of assays for the detection of fibrin(ogen)olysis based on COOH-terminal A alpha chain epitopes.

The COOH-terminal two-thirds of the fibrinogen A alpha chain is a substrate for both factor XIIIa and plasmin and is, therefore, a source of structural markers for the clinical detection of fibrin(ogen)olysis. Monoclonal antibodies (MoAbs) that bind to epitopes within this region (F-102, A alpha 563-578; F-103, A alpha 259-276) have been applied towards the development of two sensitive and specific enzyme-linked immunosorbent assays (ELISAs). The first assay, a capture (F-102)-tag (F-103) ELISA, measures plasma fibrinogen molecules whose A alpha chains are intact. The second assay, a solution phase competitive ELISA based on MoAb F-102, quantifies circulating COOH-terminal A alpha chain degradation products (A alpha FDPs), among the earliest peptides released from fibrinogen during plasmin-mediated fragment X formation. This assay features a novel preliminary plasma absorption step on concanavalin A to recover A alpha FDPs (if present in the sample) in a milieu free of immunologically cross-reactive fibrinogen. Both ELISAs use highly purified fibrinogen as the assay standard for quantitation. In control plasmas, circulating A alpha FDPs accounted for less than 2% of their respective intact fibrinogen A alpha chain concentration, suggesting a physiologic low level of proteolysis occurring at the extreme COOH-terminal portion of the molecule. Plasma A alpha FDPs were elevated (2.3% to 7.8% of their respective intact fibrinogen A alpha chain concentration) in a group of plasma from patients with documented, high serum FDPs (21 to 41 micrograms/mL). Application of the two ELISAs to characterize the course of A alpha chain proteolysis during thrombolytic therapy (TIMI phase 1) indicated that A alpha FDPs were a very early marker of the lytic state (detectable 15 minutes after treatment had been initiated), and that streptokinase and recombinant tissue plasminogen activator appeared to produce significantly different A alpha chain degradation profiles.

Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer.

Paramagnetic particles coated with a monoclonal antibody to D-dimer (mAb S4) were used to isolate and concentrate D-dimer and D-dimer-containing complexes in plasma and serum. Antibody-captured material was eluted with SDS-urea buffer and examined by either SDS-polyacrylamide or submerged SDS-agarose gel electrophoresis followed by Western blotting. The protein pattern was visualized by either polyclonal antibodies to human fibrinogen or monoclonal antibodies specific for fibrinogen derivatives containing fibrinopeptide A (FpA; mAb Y18), the N-terminus of the beta-chain in fibrin (mAb 59D8) or the gamma-chains (mAb J88B). The results obtained show that paramagnetic particles coated with mAb S4 catch intact D-dimer as well as a variety of cross-linked fibrin molecules of high-molecular-weight (HMW) in plasma. The existence of HMW derivatives in fibrinaemic serum indicates that some of the HMW fibrin related material in such plasma is not clottable. Fibrinogen/fibrin monomers and some of the fibrinogen/fibrin related derivatives found in the eluates were probably non-covalently bound to the complexes caught by mAb S4 coated particles. The present technique combines the selective concentrating power of immunoparticles and the sensitivity of immunovisualization and allows rapid and direct identification of minute amounts of circulating immunoreactive fibrinogen/fibrin derivatives.

Hematologic effects of total knee arthroplasty. A prospective evaluation.

In a prospective evaluation of 41 patients (57 knees) treated with index cemented total knee arthroplasty, perioperative blood testing was performed on blood counts, fibrinogen, and fibrin degradation products (FDP). The preoperative platelet count averaged 314/nl (range, 160-502/nl), whereas postoperatively, the count nadired at an average of 173/nl (range, 60-305/nl). Fibrinogen levels increased from 304 mg/dl (range, 170-442 mg/dl) to an average high of 647 mg/dl (range, 317-1018 mg/dl). Patients were also classified according to whether they had been treated with unilateral or bilateral procedures. Postoperatively, unilateral patients had an average 32% decrease in the platelet count, compared with the 64% decrease seen in bilateral patients. Analysis of fibrin split products revealed a trend toward greater elevation of these degradation products after bilateral procedures. The hematologic changes represented evidence of activation of the coagulation and fibrinolytic systems. These changes tended to be more pronounced in patients treated with bilateral procedures, and were manifested in postoperative elevation of fibrinogen and fibrin degradation products, with reductions in the platelet count. The degree of relative thrombocytopenia raises concerns about careful evaluation of candidates for bilateral arthroplasty, especially when determining the preoperative platelet count.

Heterozygous abnormal fibrinogen Osaka III with the replacement of gamma arginine-275 by histidine has an apparently higher molecular weight gamma-chain variant.

Congenitally abnormal fibrinogen Osaka III with the replacement of gamma Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of alpha- and gamma-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal gamma-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 gamma remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Characterization of the gamma chain platelet binding site on fibrinogen fragment D.

Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400-411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400-411 peptide in inhibiting ADP-induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.