Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

Structure-activity relationship studies of flavopiridol analogues.

Cyclin dependent kinases (CDKs) along with the complementary cyclins form key regulatory checkpoint controls on the cell cycle. Flavopiridol is a synthetic flavone that shows potent and selective cyclin-dependent kinase inhibitory activity. In this paper, we report modifications of the 3-hydroxy-1-methylpiperidinyl (D ring) of flavopiridol and their effect on CDK inhibitory activity.

Generation of an Ugi library of phosphate mimic-containing compounds and identification of novel dual specific phosphatase inhibitors.

The four-component Ugi reaction was utilized to prepare a library of dipeptidic compounds in order to explore the binding requirements of the key cell cycle phosphatase, Cdc25. Several phosphate surrogates were incorporated into the Ugi product to mimic either the mono- or bis-phosphorylated substrate.

Dysidiolide and related gamma-hydroxy butenolide compounds as inhibitors of the protein tyrosine phosphatase, CDC25.

Synthetic dysidiolide, as well as several related compounds containing a gamma-hydroxybutenolide moiety, were tested in in vitro Cdc25 assays against both synthetic and natural substrates. Contrary to literature values which are in the low micromolar range, we observe only millimolar inhibitory activity for these compounds versus Cdc25 phosphatase.

An ELISA for factor X activation peptide: application to the investigation of thrombogenesis in cardiopulmonary bypass.

An ELISA for measurement of factor X activation peptide (FXAP) in plasma has been developed. The capture antibody was generated by immunization with a carrier-coupled synthetic peptide based on the amino acid sequence of the C terminal region of native human FXAP: the tag antibody was a commercial polyclonal antibody to factor X. Because of limited specificity of the capture antibody to FXAP compared with factor X, a plasma processing step precipitated plasma factor X and also permitted a concentration step, enabling detection of FXAP below the lower limit of the normal range in plasma. The overall intra- and inter-assay coefficients of variation were approximately 5% and approximately 11%, respectively. 18 normal laboratory control subjects had FXAP levels of 2.12 +/- 0.82 ng/ml (mean +/- SEM). Eight patients undergoing surgery and cardiopulmonary bypass progressively generated FXAP throughout the surgery with mean FXAP rising to 11.73 +/- 4.66 ng/ml, and this resulted in increased generation of thrombin detected by measurement of plasma levels of F1 + 2. Levels of FXAP rose significantly ahead of those of factor IX activation peptide (FIXAP), supporting a suggestion that contact system activation can not be the primary stimulus to coagulation in bypass. The ELISA to FXAP will be useful in the study of mechanisms of thrombogenesis in clinical situations where the coagulation system is activated.

Thrombogenic mechanisms in the human: fresh insights obtained by immunodiagnostic studies of coagulation markers.

Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, meaningful in vivo studies of thrombogenic mechanisms have previously been hindered by the absence of suitable assays. This article reviews the recent development and/or contemporary clinical application of plasma-based immunoassays for coagulation markers (factor XIIa, factor IX activation peptide, prothrombin fragment F1 + 2, thrombin-antithrombin complex and fibrinopeptide A) and for the fibrinolytic marker, D-dimer, which have enabled a critical re-appraisal of some long-standing hypotheses. In chronic renal disease the intrinsic coagulation pathway was found to be activated before haemodialysis and increased end-stage coagulation activity was detected during dialysis when heparinization was limiting. No evidence was found to support the generally accepted hypothesis that thrombogenesis in dialysis is triggered by stimulation of the contact system following exposure of blood to the dialyser membrane. Instead, it is postulated that it is a failure of regulation of end-stage coagulation proteinases (owing to the absence of endothelium) which is responsible for increased thrombogenesis in the dialyser circuit. Excessive end-stage coagulation activity was observed during cardiopulmonary bypass (CPB) surgery and in patients undergoing general thoracic surgery. The data did not accord with the hypothesis that the contact system provides the major thrombogenic trigger in CPB surgery. It is proposed that, in general thoracic surgery, a powerful procoagulant stimulus is provided via the tissue factor-factor VIIa pathway and that the same mechanism is also primarily responsible for triggering thrombogenesis during CPB surgery. The established hypothesis of a prethrombotic state in hereditary AT III deficiency is challenged by the inability to detect increased coagulation activity in asymptomatic AT III deficient patients. It is concluded that the AT III concentration in deficient members is sufficient to enable regulation of the coagulation system in the basal state, whereas failure to regulate the coagulation system only occurs following a major procoagulant stimulus, which overwhelms the impaired inhibitory capacity and triggers thrombosis. These findings highlight the advantages of using plasma-based immunoassays to investigate thrombogenic mechanisms in hypercoagulable states and have important implications for the further study and treatment of blood-surface interactions and thrombotic disease.

Mechanisms of thrombin generation during surgery and cardiopulmonary bypass.

Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, the mechanisms involved during the in vivo response to hypercoagulable stimuli are still unclear. We have used plasma-based enzyme-linked immunosorbent assays (ELISAs) to study the mechanisms by which the coagulation system is activated in vivo during human cardiopulmonary bypass (CPB) surgery (n = 8). A novel immunoassay for factor XIIa was used to detect activation of the contact system, factor IX activation peptide (FIXAP) was used as a marker for activation of factor IX, and prothrombin fragment F1 + 2 (F1 + 2) was used as a marker for thrombin generation. The ELISA for FIXAP is described for the first time herein. F1 + 2 levels increased early in response to surgical intervention: from a baseline of 38.7 +/- 9.7 ng/mL (mean +/-SE), levels increased rapidly during surgery and bypass to a maximum of 448.5 +/- 92.0 ng/mL. A modest yet significant increase in factor XIIa levels from 3.47 +/- 0.54 ng/mL to 4.33 +/- 0.85 ng/mL was evident during surgery before bypass, but no further significant increase was detected on establishing extracorporeal circulation. FIXAP levels demonstrated a small and late increase during surgery from 4.98 +/- 0.55 ng/mL to a maximum of 10.20 +/- 1.23 ng/mL, the increase beginning at the time of near maximal F1 + 2 levels. There was no association between activation of the contact system (factor XIIa levels) and the generation of thrombin (F1 + 2 levels). However, a strong association (r = .705) was apparent between the generation of thrombin (F1 + 2 levels) and activation of factor IX (FIXAP levels), despite the delay between the activation of prothrombin and factor IX. The data do not support the established view that contact activation resulting from exposure of blood to foreign surfaces is the major procoagulant stimulus in CPB. Instead, the results suggest that the main trigger to coagulation during CPB surgery was provided via the tissue factor-factor VIIa mechanism in response to the cutting of blood vessels, which directly activated factor X and then prothrombin. The late activation of factor IX, which presumably also contributed to maximal prothrombin activation, could have arisen due to direct tissue factor-factor VIIa action, or by secondary feedback action of thrombin on the intrinsic system.

Thrombin production, inactivation and expression during open heart surgery measured by assays for activation fragments including a new ELISA for prothrombin fragment F1 + 2.

Activation of coagulation was studied during the peri-operative period in patients undergoing cardiopulmonary bypass (CPB) surgery using activation markers which have recently become available: prothrombin fragment F1 + 2 (F1 + 2), which is a measure of total thrombin generation, and thrombin-antithrombin complex, which is a measure of inactivation of free thrombin by antithrombin. Levels of the specific marker of fibrin breakdown, D-dimer, were also determined. F1 + 2 levels were assessed using a newly developed ELISA described herein which employs a neoantigen-specific capture antibody raised using a synthetic peptide; the latter antibody has been pre-adsorbed against prothrombin to ensure high specificity for F1 + 2. Increased generation of thrombin during surgery was clearly demonstrated despite maintenance of a high concentration of heparin during the period of extracorporeal blood circulation. There was a close association (r = 0.882) between the generation of thrombin (F1 + 2 levels) and its inhibition (TAT levels). Differences were noted, however, between the information provided by F1 + 2 and TAT, which are interpreted with regard to the different in vivo fates of F1 + 2 and thrombin. The enhanced activation and inhibition of coagulation observed during CPB was suppressed once physiological blood circulation was restored, with F1 + 2 returning to pre-surgical levels within 24 h after surgery. During the post-operative period D-dimer levels, which rose in concert with F1 + 2 and TAT levels, remained highly elevated, suggesting that not all of the generated thrombin was inactivated by antithrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

A microtitre plate ELISA to measure thrombin-antithrombin complex using pan-specific antibodies.

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed to measure plasma levels of thrombin-antithrombin complex (TAT). The assay is performed in a microtitre plate using polyclonal antibodies specific for antigenic determinants on prothrombin and antithrombin. Antibody to prothrombin was immobilized on a solid phase, using a titre predetermined to optimize capture of TAT. The performance of the microtitre plate ELISA for TAT has been extensively investigated and compared with the performance characteristics of a tube-based ELISA for TAT which is available commercially (Enzygnost-TAT, from Behringwerke, Marburg, Germany). Studies with plasma containing various levels of prothrombin showed that the zymogen competed with TAT for capture antibody in both assays. Variations in prothrombin levels between plasma samples present a potential source of artifact, but one which does not critically affect the performance of either assay in detecting large elevations in TAT. A high correlation (r = 0.88) was established between the results of plasma samples assayed by both assays, whether citrate or EDTA anticoagulant was used to prepare plasma. High correlations (r > 0.90) were also established for each assay between the results of plasma prepared with EDTA as compared to citrate anticoagulant. Both assays were able to discriminate completely between a group of 16 normal controls and a group of 31 patients with disseminated intravascular coagulation (DIC).

Hemodialysis and heparin. Alternative methods of measuring heparin and of detecting activation of coagulation.

A bolus dose of heparin was administered pre-dialysis to patients (n = 6) undergoing regular maintenance hemodialysis with cuprophane flat plate and hollow fiber membranes. Blood samples were withdrawn at hourly internals for measurement of a) heparin and b) activation markers of coagulation, fibrinolysis and platelets. Two assay methods for heparin were employed; amidolytic assay of anti-factor Xa activity in plasma and a simple whole blood clotting time based upon factor Xa inhibition (Heptest). Results from these heparin assays correlated well with each other (r = 0.89) and both showed similar negative correlations (r = -0.72, amidolytic and r = -0.66, Heptest) with levels of a marker of fibrin clot formation, fibrinopeptide A (FPA). Large differences in levels of FPA were observed during dialysis with the two dialyzer types, when similar levels of heparin were present. Heparin levels declined from 1-5-h dialysis and were associated with rises in plasma levels of FPA, thrombin-antithrombin complex (TAT) and beta thromboglobulin (BTG), but not of D-dimer. Regression analysis revealed the best correlation was between FPA and TAT (r = 0.94), followed by FPA and BTG (r = 0.81). FPA and D-dimer exhibited significant, but lower (r = 0.42), correlation. TAT levels, like FPA levels, showed good correlation with heparin (r greater than 0.65). It is concluded that the Heptest assay may be a useful bedside measurement of heparin levels and the TAT assay may be a simplified means of evaluating coagulation system activation during dialysis.

A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis: thrombin-antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen.

Measurements were made of levels of D-dimer in plasma and serum, thrombin-antithrombin complex (TAT) in plasma and fibrinogen/fibrin fragment E antigen (FgE) in serum in a normal healthy control group and in patients with a range of disorders associated with hypercoagulability. Levels were determined in 31 normal healthy controls, 30 patients with disseminated intravascular coagulation (DIC), 21 patients with deep venous thrombosis (DVT), 27 patients with myocardial infarction (MI), 26 patients with acute leukaemia and 56 patients with liver disease. Considering all subjects, significant correlations were established between the results of all assays. Notably high correlations (r greater than 0.9) were established between plasma and serum levels of D-dimer, between plasma levels of D-dimer and serum levels of FgE, and between serum levels of D-dimer and FgE. All assays showed very high discrimination (sensitivity) between the normal control group and patients with DIC (97-100%), but there were marked differences between the assays in sensitivity for DVT and MI. In general, the FgE assay was more sensitive than the D-dimer assay, whilst both the FgE and D-dimer assays were more sensitive than the TAT assay. The same trends were apparent in the capability of the assays to discriminate between the normal control group and patients with acute leukaemia and liver disease: disorders with an unknown prevalence of activation of coagulation/fibrinolysis. Our results indicated that measurements of fibrinogen/fibrin degradation products (FDPs) in serum were almost unaffected by artefacts. The data further suggested that the broad-spectrum FgE assay was better than the more specific D-dimer assay in detecting clinical hypercoagulability. Our study showed that, in the clinical conditions examined, FDPs were more effective markers of hypercoagulability than TAT.

Assessment of hypercoagulable states by measurement of activation fragments and peptides.

Broad spectrum assays which measure a range of fibrinogen/fibrin derivatives (FDPs) in serum have become an established means of identifying activation of blood coagulation and/or fibrinolysis, such as occurs in disseminated intravascular coagulation (DIC). There is considerable interest in the application of these assays to the diagnosis of other hypercoagulable states, such as recurrent deep venous thrombosis and myocardial infarction. In recent years, more sensitive and specific FDP assays (e.g. for fragment E, fragment E neoantigen, D-dimer, fragment D neoantigen, fibrinopeptide A and fibrin fragment beta 15-42) have been devised, some of which allow measurement in plasma of FDPs without interference from fibrinogen or certain of its derivatives. It was predicted that these assays would both avoid the possibility of artifacts introduced as a consequence of serum preparation and improve detection of hypercoagulable states. In the light of these expectations we have reviewed data published on the use of assays to detect clinical hypercoagulability, giving prominence to assays of crosslinked fibrin derivatives and nothing particularly certain studies that have compared the performance of different assays on the same samples. The accumulating evidence indicates that all of the assays are adequate for detection of DIC. The same cannot be said for other hypercoagulable states. Here much variation is evident between different studies of similar patients in the ability of a particular marker to discriminate between a normal control group and patients determined to be hypercoagulable by an independent method. This variability would seem to be a function of patient group heterogeneity and selection, as assays that detect different antigenic determinants produce results on the same plasma samples that are well correlated. It appears that the precise antigenic determinant does not critically affect detection of hypercoagulability. Additionally, some studies have indicated that use of serum need not introduce artifacts. Despite there being no other obvious advantage, the convenience of some of the plasma assays may well encourage their widespread use. Assays have also been developed for measuring activation fragments of coagulation proteins (e.g. prothrombin fragment F1 + 2 and protein C activation peptide) and for proteinase inhibitor complexes (e.g. thrombin-antithrombin complex) generated during activation of coagulation. The latter assays have been useful in providing a biochemical definition of a 'prethrombotic state'.

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes.

Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. The midpoint redox potential of the cytochrome is 100-130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5-7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. The Nernst plots of titration data have slopes of 75-115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4-6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.