Characterization of cleaved plasma protein S with a monoclonal antibody-based assay.

A monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i.e. the carboxy-terminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.

Identification of protein C epitopes altered during its nanoencapsulation.

Protein C is a plasmatic inhibitor which regulates the blood coagulation mechanism by modulating the anticoagulant response. The improvement of its bioavailability would be beneficial for the treatment of the disorders caused by its homozygous deficiency or by an other plasmatic inhibitor deficiency. In this context, the protein C encapsulation into biodegradable nanoparticles could be used to approach the problem. However, the method used to prepare the nanoparticles requires the use of ultrasonication and of an organic solvent such as methylene chloride which interferes with protein activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that neither ultrasonication nor methylene chloride, singly or in combination, led to protein C aggregation or cleavage. Thus, a binding study using an ELISA assay with four characterized monoclonal antibodies was carried out to identify the epitopes damaged by these experimental constraints. The correlation between the immunological assay and a functional one i.e. by the means of the assay of its anticoagulant activity (activated partial thromboplastin time) made it possible to show that protein C amino acids 166-169 of the activation peptide were probably altered after ultrasonication and methylene chloride treatment. Indeed, it is likely that these residues were no longer surface-exposed but had moved inside the protein core.

A monoclonal antibody recognizing the activation domain of protein C in its calcium-free conformation.

A monoclonal antibody (mAb) binding to protein C (PC) heavy chain but not to activated PC was found to inhibit PC activation by free thrombin, suggesting that epitope involved the activation site. Using a set of overlapping synthetic peptides, we confirmed that this mAb recognizes the sequence encompassing the thrombin cleavage site (165QVDPRLI(171)). Surprisingly, epitope was only accessible in the absence of calcium, half-maximal inhibition of mAb binding occurring at 100 microM Ca2+. Thus, our antibody provides direct evidence that conformation and/or accessibility of the activation site differ between the apo and Ca2+-stabilized conformers of PC.

Evaluation of a new automated ELISA test for von Willebrand factor using two monoclonal antibodies.

A new automated quantitative enzyme linked immunosorbent assay (ELISA) for the detection of human von Willebrand factor (vWF), VIDAS vWF, has been developed for use on the VIDAS analyser (bioMérieux). The two-step capture/tag test relies on two monoclonal antibodies (mAbs), the second one being labelled with alkaline phosphatase. The lower limit of detection of the assay is < 1 IU/dl, and the upper limit of detection is 120 IU/dl. There is no hook effect for concentrations up to 1100 IU/dl. Intra- and inter-assay precision ranges from 3% and 5%. Assays are performed preferentially using citrated plasma and in these conditions the 95% confidence intervals for normal values are 52-154 IU/dl and 60-200 IU/dl for O blood group and non-O blood group subjects, respectively. Using the lower limits of the normal range as the cut-off level, all patients tested with type 1 (n = 29) or 3 (n = 2) von Willebrand disease (vWD) would be accurately classified with the new ELISA. Comparing the VIDAS test with a conventional ELISA gave a good correlation and comparable results with type 1 and 3 vWD patients (n = 31; r = 0.99; y = 0.99x + 0.24), type 2A and 2B vWD patients (n = 20; r = 0.99; y = 1.05x-1.65) and normal subjects (n = 204; r = 0.94; y = 1.06x-2.6).

VIDAS D-dimer: fast quantitative ELISA for measuring D-dimer in plasma.

VIDAS D-dimer (bioMérieux) is a new quantitative ELISA for D-dimer determination designed for the VIDAS automated system. The test contains single-dose, ready-to-use reagents and is completed within 35 min. Quantitative results are obtained from a calibration curve stored in the software of the system and expressed as fibrinogen equivalent units. The two-step capture/tag test relies on two complementary monoclonal anti-D-dimer antibodies, the second one being labeled with alkaline phosphatase. The upper limit of the measuring range is 1000 micrograms/L and the lower detection limit is <50 micrograms/L, which is below the lower limit of the reference interval (68-494 micrograms/L). Reproducibility (CV) within and between runs ranges from 5% to 7%. There is no interference from heparin, bilirubin, hemoglobin, fibrinogen degradation products, or plasma turbidity. Comparison with a conventional ELISA (y) gave good correlation (r= 0.91, n= 579) and comparable results (y= 1.35x - 148, S(y/x)= 750), especially for D-dimer concentrations ranging from 0 to 1000 micrograms/L (y= 1.09x - 10.6, r= 0.88, S(y/x)= 170).

Evaluation of a new rapid quantitative D-dimer assay in patients with clinically suspected deep vein thrombosis.

The sensitivity and specificity for deep vein thrombosis (DVT) of a new rapid, quantitative and precise (total imprecision < 10%) D-dimer assay suitable for individual measurements (VIDAS D-DIMER, bio-Mérieux, France) were evaluated in a consecutive series of 103 in- and out-patients submitted to serial compression ultrasonography (C-US) for the clinical suspicion of DVT (n = 66) or of DVT recurrence (n = 37) and symptoms lasting from 1 to 15 days. DVT was found in 22 patients at baseline testing and no patient with an initially negative C-US developed vein incompressibility at follow up. The time elapsed from the onset of symptoms was negatively associated with D-dimer levels both in patients with and in those without DVT. In the entire series of patients, the sensitivity of a positive D-dimer test ( > or = 1.0 microgram/ml) for the presence of DVT was 96% (21/22 patients, 95% confidence interval 75-100%) with a specificity of 75% (64-84%), a negative predictive value of 98% (90-100%), a positive predictive value of 51% (35-67%), and an overall accuracy of 80% (70-87%). A normal D-dimer value (0.22 microgram/ml) was observed in one patient with DVT and symptoms lasting from 15 days. The approach of withholding C-US testing in patients with symptoms lasting from less than 11 days and D-dimer levels below the cut-off value was compared to serial C-US testing alone in a cost-effectiveness analysis subdividing the 66 patients with a first episode according to their clinical pretest probability of DVT. Thrombosis was detected in 6.7% of the patients in the low probability group (n = 15), 16.7% of the patients in the moderate probability group (n = 24), 51.9% of the patients in the high probability group (n = 27) and 8.1% of patients with suspected DVT recurrence. Calculated cost-savings for each DVT diagnosed ranged from 5% in the high pretest probability group to 55% in the low pretest probability group and to 77% in patients with suspected DVT recurrence. The safety of avoiding C-US testing in symptomatic patients with a negative D-dimer test should be evaluated in clinical management studies.

Contribution of a new, rapid, individual and quantitative automated D-dimer ELISA to exclude pulmonary embolism.

The performance of a new automated ELISA for a rapid, individual and quantitative measurement of plasma D-dimer (VIDAS D-dimer) has been evaluated. First, a study of 100 patients was performed in order to choose the best couple of antibodies in comparison with an already clinically validated ELISA. Then the results were certified in a prospective study including 195 consecutive patients suspected of pulmonary embolism (PE). For a cut-off level of 500 ng/ml VIDAS D-dimer showed a sensitivity of 100% (95% confidence interval 92-100), a specificity of 37.6%, a negative predictive value of 100% (95% CI 93.3-100) and a positive predictive value of 33.1%. During a 6 months' follow-up no patient (95% CI 0-6.4) with D-dimer < 500 ng/ml presented a new suspicion of venous thromboembolic disease. These results suggest that this rapid and single-dose ELISA provides a very useful tool for the clinician to exclude on a day-to-day basis the diagnosis of PE.

Amino acids 225-235** of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex.

Protein C (PC) is a vitamin K-dependent zymogen that inactivates factors Va and VIIIa after its activation by thrombin complexed to thrombomodulin. We characterized a monoclonal antibody (mAb) against PC, whose only influence on PC functions was to inhibit PC activation by the thrombin-thrombomodulin complex. It recognized an epitope in the PC heavy chain, the conformation of which is calcium-dependent. The mAb did not recognize a natural PC variant that was not activated by the thrombin-thrombomodulin complex (mutation R229Q) and did recognize a synthetic peptide corresponding to PC amino acids 225-235 in an Elisa assay. The peptide inhibited PC activation by the thrombin-thrombomodulin complex. These data confirm that the calcium-binding loop of the serine-protease domain is involved in the interaction of PC with the thrombin-thrombomodulin complex.

A new assay based on thrombin generation inhibition to detect both protein C and protein S deficiencies in plasma.

Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.

Soluble uridine diphospho-D-glucose: mycosporin glucosyltransferase from spores of Ascochyta fabae Speg.

The enzyme properties of a soluble uridine 5'-diphosphate (UDP) glucose: mycosporin-2 glucosyltransferase from spores of Ascochyta fabae Speg. (Fungi imperfecti) were studied. The optimal conditions for the glucose transfer from UDP-glucose to the mycosporin-2 (the amide form being the best acceptor) were determined; for maximal activity the glucosyltransferase requires a pH of about 8.5 and the presence of divalent cations (Mn(2+) being more efficient than Ca(2+) or Mg(2+)). The reaction was not reversible in presence of large amounts of UDP.