Emicizumab mimics the hemostatic activity of activated factor VIII (FVIIIa) within the tenase complex. Despite functional similarities between FVIIIa and emicizumab, conventional laboratory methods ...designed for monitoring of FVIII activity are inappropriate for the measurement of emicizumab. At present, a modified one stage (FVIII) assay (mOSA) is mainly used for emicizumab monitoring. Two-stage chromogenic FVIII assays based on human factors can be used, although limited performance due to lack of corresponding optimization might be observed. Furthermore, the presence of FVIII or anticoagulants in the patient sample may falsify assay results. To address these issues, we optimized and evaluated a two-stage chromogenic assay (emi-tenase) for measurement of emicizumab in plasma samples. Heat inactivation of samples was established to abolish the influence of endogenous or substituted FVIII. The lower limit of quantification (LLoQ) was found to be 2 μg/ml in a manual assay format and 9.5 μg/ml on an automated coagulation analyzer. Intra- and inter-assay coefficients of variation (CV) did not exceed 20%. Analysis of 17 patient plasma samples with severe haemophilia A under emicizumab treatment showed good correlation of results between the emi-tenase assay and the mOSA (Cohens Kappa coefficient = 0.9). Taken together, the emi-tenase assay allows specific measurement of emicizumab plasma levels over a broad concentration range (10 μg/ml to 100 μg/ml). The assay can be applied on an automated coagulation analyzer, demonstrating its applicability within a routine laboratory setting.
Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we ...present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca
-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca
ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.
Schematic illustration of the TCL-SPION-Apt bioconjugate system (Wang et al., 2008).
Every year a large number of new cases of colorectal cancer are diagnosed in the world. Application of Epirubicin ...(Epi) in treatment of cancer has been limited due to its cardiotoxicity. Specific delivery of chemotherapy drugs is an important factor in reducing the side effects of drugs used in chemotherapy. Enhanced permeability, retention effect and magnetic resonance (MR) traceability of super paramagnetic iron oxide nanoparticles (SPION) make them a great candidate in cancer therapy and imaging. In this study, Epirubicin-5TR1 aptamer–SPION tertiary complex was evaluated for the imaging and treatment of murine colon carcinoma cells (C26 cells, target). For cytotoxic studies (MTT assay), C26 and CHO-K1 (Chinese hamster ovary cells, nontarget) cells were treated with either Epi or Epi–Apt–SPION tertiary complex. Internalization was evaluated by flow cytometry. Finally, Apt-SPION bioconjugate was used for imaging of cancer in vivo. Flow cytometric analysis showed that the tertiary complex was internalized effectively to C26 cells, but not to CHO-K1 cells. Cytotoxicity of Epi–Apt–SPION tertiary complex also confirmed internalization data. The complex was less cytotoxic in CHO-K1 cells when compared to Epi alone. No significant change in viability between Epi- and complex-treated C26 cells was observed. Magnetic resonance imaging (MRI) indicated a high level of accumulation of the nano-magnets within the tumor site. In conclusion Epi–Apt–SPION tertiary complex is introduced as an effective system for targeted delivery of Epi to C26 cells. Moreover this complex could efficiently detect tumors when analyzed by MRI and inhibit tumor growth in vivo.
Direct oral anticoagulants (DOACs) apixaban and rivaroxaban are broadly used in the management of venous thromboembolism (VTE). Although not routinely required, measurement of their plasma ...concentration is advised for an increasing number of indications. Due to the lack of therapeutic ranges, current guidelines recommend reporting DOAC plasma levels together with expected levels from previous pivotal studies. The aim of this study was to assess DOAC level variation in a large VTE patient population. Drug concentrations determined by measurement of the anti-Xa-activity using drug-specific calibrators in citrated plasma samples from patients on rivaroxaban (n = 1471) or apixaban (n = 725) were analyzed. Observed 5th-95th percentile ranges of apixaban peak/trough levels (63-299/13-114 ng/mL for 5 mg, 37-161/7-68 ng/mL for 2.5 mg twice daily) were similar to previously reported mass-spectrometry-based reference data, and 10th-90th percentile ranges of rivaroxaban peak/trough levels (98-367/8-55 ng/mL for 20 mg, 51-211/5-27 ng/mL for 10 mg once daily) were even narrower. Age and drug levels correlated weakly (r ≤ 0.330). Drug levels measured repeatedly in subgroups of patients showed a strong correlation (r ≥ 0.773). In conclusion, anti-Xa-activity-based measurement of apixaban and rivaroxaban yields reliable results. However, the paucity of levels off-range underlines the need for evidence-based thresholds to better assist clinical decision making.
Activated protein C (APC) is a critical regulator of thrombin formation and thereby protects against thrombosis. On the other hand, overwhelming formation of APC increases the risk of bleeding such ...as in trauma-induced coagulopathy. Thus, pharmacological inhibition of APC activity may improve blood clottability in certain clinical situations. In this study, we demonstrate that the DNA aptamer HS02-52G binds with fast onset (1.118 ± 0.013 × 10
M
s
) to APC and possesses a long residence time of 13.5 min within the aptamer-APC complex. Functional analysis revealed HS02-52G as a highly potent and specific inhibitor of APC in plasma and whole blood with IC
values ≤30 nM, whose activity can be readily neutralized by the short complementary DNA molecule AD22. These features qualify the novel aptamer-antidote pair as a candidate treatment option for acute APC-related bleedings.
Elevated D-dimer levels during anticoagulant therapy with vitamin K antagonists (VKA) are associated with an increased risk of thrombosis. It has been hypothesized that elevated D-dimer levels in ...patients receiving direct oral anticoagulants (DOACs) also indicate an increased risk of thrombosis recurrence, but data on the distribution of D-dimer levels in patients with VTE on DOACs are sparse. In the present study we retrospectively analyzed D-dimer levels in patients taking DOACs after first or recurrent venous thrombosis (
= 1,716, 1,126 thereof rivaroxaban, 481 apixaban, 62 edoxaban, and 47 dabigatran). Patients on VKA (
= 402) served as control group. Thrombotic events in the study population were categorized into distal deep venous thrombosis (DVT,
= 552 patients), distal DVT with pulmonary embolism (PE,
= 166), proximal DVT (
= 685), proximal DVT with PE (
= 462), PE without DVT (
= 522), DVT of the upper extremity (
= 78), cerebral venous sinus thrombosis (CVST,
= 48), and other venous thrombosis (
= 74). In VKA users a median D-dimer level of 0.20 mg/l was observed. In patients on DOACs D-dimer levels were significantly higher, with 0.26 mg/l for rivaroxaban, 0.31 mg/l for apixaban (
< 10
each), 0.24 mg/l for edoxaban (
= 2 × 10
), and 0.25 mg/l for dabigatran (
= 4 × 10
). These differences in comparison to patients on VKA treatment could not be explained by the patients' age, sex, body mass index, and type of thrombosis as these characteristics did not differ significantly between cohorts. Moreover, the prevalence of D-dimer levels above age-adjusted cut-offs ≥0.50 mg/l in ≤50-year-old patients, ≥(age × 0.01) mg/l in >50-year-old patients was higher in patients on rivaroxaban (13.9%, RR 1.74, 95% CI 1.21-2.50), apixaban (17.0%, RR 2.14, 95% CI 1.45-3.15) and dabigatran (23.4%, RR 2.94, 95% CI 1.59-5.44) than in patients on VKA (8.0%). In patients on edoxaban D-dimer levels above the reference range were observed in 14.5%, but no statistical significance was reached in comparison to the VKA cohort. In conclusion, the obtained data suggest, that the type of oral anticoagulant should be considered in the clinical assessment of D-dimer levels in thrombosis patients. Further studies are warranted to evaluate a potential association between elevated D-dimer levels and thrombosis risk in patients on DOACs.
A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) is a metalloprotease that regulates the size of circulating von Willebrand factor (vWF) multimers. Severe ...lack of ADAMTS-13 activity <10% of normal (0.1 IU/mL) leads to thrombotic thrombocytopenic purpura (TTP), a specific type of thrombotic microangiopathy (TMA). Timely determination of plasma ADAMTS-13 activity is essential to discriminate TTP from other types of TMA with respect to adequate treatment. Identification of the minimal substrate motif for ADAMTS-13 within the A2 domain of vWF (vWF73) as well as the generation of monoclonal antibodies (mAbs) that specifically recognize the ADAMTS-13 cleavage site enabled the development of a variety of methods for determination of plasma ADAMTS-13 activity. In order to further extend the range of analytical platforms applicable for quantitative determination of plasma ADAMTS-13 activity, a specific, vWF/mAb-based assay with flow cytometric readout was developed and validated. Basic assay characteristics include a total assay time of 80 to 90 min, a near linear dynamic range from 0.005 (lower limit of quantification) to 0.2 IU/mL, and intra- and interassay coefficients of variation below 5 and 30% at input plasma ADAMTS-13 activities of 0.015 and ≤0.050 IU/mL, respectively. When compared to the results obtained with a commercially available quantitative ADAMTS-13 activity ELISA, analysis of 18 plasma samples obtained from patients with suspected TTP revealed full agreement of results with respect to the clinical 0.1 IU/mL TTP threshold. Based on these data, it is assumed that the described assay principle can be successfully transferred to virtually all laboratories that have a flow cytometer available.
Coagulation factor XIII (FXIII) is a protransglutaminase which plays an important role in clot stabilization and composition by cross-linking the α- and γ-chains of fibrin and increasing the ...resistance of the clot to mechanical and proteolytic challenges. In this study, we selected six DNA aptamers specific for activated FXIII (FXIIIa) and investigated the functional characterization of FXIIIa after aptamer binding. One of these aptamers, named FA12, efficiently captures FXIIIa even in the presence of zymogenic FXIII subunits. Furthermore, this aptamer inhibits the incorporation of FXIII and α2-antiplasmin (α2AP) into fibrin(ogen) with IC
-values of 38 nM and 17 nM, respectively. In addition to FA12, also another aptamer, FA2, demonstrated significant effects in plasma-based thromboelastometry (rotational thromboelastometry analysis, ROTEM)-analysis where spiking of the aptamers into plasma decreased clot stiffness and elasticity (
< 0.0001). The structure-function correlations determined by combining modeling/docking strategies with quantitative in vitro assays revealed spatial overlap of the FA12 binding site with the binding sites of two FXIII substrates, fibrinogen and α2AP, while FA2 binding sites only overlap those of fibrinogen. Taken together, these features especially render the aptamer FA12 as an interesting candidate molecule for the development of FXIIIa-targeting therapeutic strategies and diagnostic assays.
Capillary electrophoresis-based SELEX (CE-SELEX) is an efficient technique for the isolation of aptamers binding to a wide range of target molecules. CE-SELEX has a number of advantages over ...conventional SELEX procedures such as the selection of aptamers can be performed on non-immobilized targets, usually within a fewer number of selection cycles. Here we describe a complete procedure of CE-SELEX using activated protein C (APC) as the target protein.
PDF
