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Abstract: Introduction: The direct thrombin inhibitor dabigatran interferes with thrombophilia screening and with the diagnosis of hemostasis disorders that develop during treatment with the anticoagulant. In vitro addition of idarucizumab, a humanized antibody fragment that binds dabigatran, to plasma samples containing dabigatran fully neutralizes the drug. This study was carried out to determine whether binding of dabigatran on selected insoluble commercial adsorbent material, DOAC‐STOPR, was as efficient as idarucizumab to neutralize the anticoagulant activity of the drug in vitro. Methods: Coagulation assays sensitive to dabigatran were carried out with patient and control plasma samples spiked with dabigatran and supplemented with idarucizumab or incubated with adsorbent material. Results: In samples containing upto 10 000 ng/mL dabigatran, the adsorption procedure was at least as efficient as the addition of idarucizumab to neutralize the activity of the anticoagulant drug. Neither the adsorption procedure nor the addition of idarucizumab did impair routine coagulation assays carried out with plasma devoid of dabigatran, such as the activated partial thromboplastin time, prothrombin time, fibrinogen Clauss, and the thrombophilia screening assays used to detect antiphospholipid antibodies or activated protein C resistance. In addition, the adsorption procedure did not interfere with the detection of lupus anticoagulant samples. Conclusions: Adsorption of dabigatran in plasma samples containing the drug neutralizes its activity as efficiently as the addition of idarucizumab. This method allows the evaluation of thrombophilia markers without interruption of anticoagulation therapy or the detection of hemostasis disorders in patients treated with the drug. [ABSTRACT FROM AUTHOR]

Introduction: Postinfusion ReFacto AF levels can be difficult to measure accurately due to discrepancies between one‐stage and chromogenic FVIII assays. To overcome this, the use of the ReFacto AF laboratory standard (RAFLS) is recommended, but there are discordant reports regarding its usefulness. Aim: We investigated whether calibration with RAFLS and measurement of ReFacto AF levels are influenced by the choice of reagents and patient‐specific factors in one‐stage FVIII assays. Methods: Calibration curves were generated with both the RAFLS and a plasma standard using different F8DPs and one‐stage FVIII assay reagents. This selection of reagents was then used to determine FVIII levels in the plasma of patients repeatedly treated with ReFacto AF. Results were compared with those obtained with a chromogenic assay. Results: F8DP devoid of von Willebrand factor (VWF) falsely increased the values of RAFLS pro‐coagulant activity generated using the APTT reagent. The resulting RAFLS calibration curve underestimated ReFacto AF levels to be half of their true concentration. The use of RAFLS with F8DP containing VWF reduced the discrepancy observed between the one‐stage and chromogenic FVIII assays. However, the mean difference between the two assays still varied up to 50% depending on the patient. Conclusions: The RAFLS is a suitable calibrator for one‐stage FVIII assays carried out with F8DP containing VWF. However, calibration with the RAFLS does not avoid the effect of patient‐specific variables that contribute to discrepancies between the measurements of ReFacto AF levels with one‐stage and chromogenic FVIII assays. [ABSTRACT FROM AUTHOR]

Introduction: Acquired thrombotic thrombocytopenic purpura is a rare disease associated with the production of autoantibodies against the VWF-cleaving protease ADAMTS13. The detection of these antibodies is made difficult by the instability of ADAMTS13 in citrated plasma and the time-consuming ADAMTS13 assays. The aim of our study was to evaluate the optimal conditions for detecting anti-ADAMTS13 inhibitory antibodies with the novel automated chemiluminescent immunoassay HemosIL R AcuStar ADAMTS13 Activity assay., Methods: The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2 hours incubation at 25°C, 37°C, or 37°C after addition of Ca 2+ to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosIL R AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the Technozym R ADAMTS13 activity ELISA., Results: The criterion of parallelism was respected for the 3 incubation methods over the range of ADAMTS13 concentrations relevant for the detection of ADAMTS13 inhibitor antibodies in a Bethesda assay. In agreement with this observation, all the incubation methods permitted the accurate detection and quantification of inhibitory anti-ADAMTS13 antibodies in the samples from patients with acquired thrombotic thrombocytopenic purpura., Conclusion: Incubation of plasma samples with normal plasma at 25°C, 37°C, or 37°C after addition of Ca 2+ can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosIL R AcuStar ADAMTS13 Activity assay., (© 2020 John Wiley & Sons Ltd.)

Introduction: The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs., Aim: The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels., Methods: We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate ® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta ® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla ® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism., Results: Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25 IU/dL in spiked samples and in detection of FVIII activity (≥1 IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results., Conclusion: Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments., (© 2018 John Wiley & Sons Ltd.)

SY 161-P5, a polyethylene glycol derivatized (PEGylated) mutant of the recombinant Staphylokinase (rSak) variant SakSTAR, exhibiting reduced antigenicity is in clinical development for treatment of acute myocardial infarction as a single bolus injection. A series of safety studies were performed in vivo as a routine toxicology program with SY 161-P5 (PEG-rSakSTAR) and with the recombinant Staphylokinase variant Sak42D (rSak42D). For both compounds, intravenous single bolus injections of up to 100-fold therapeutic equivalent, as well as repeated injections during 7 to 28 days revealed no significant pathological findings in mice, rats or hamsters. However, New Zealand white rabbits developed clinically silent, multifocal myocarditis following single or repeat doses of SY 161-P5 or of Sak42D. These findings were dose-independent and reversible. A similar species-specific cardiotoxic effect has previously been described for other proteolytic proteins, including the approved drugs Streptokinase and Acetylated Plasminogen Streptokinase Complex (APSAC). The large experience with these drugs, as well as the clinical data accumulated both with PEGylated and non-PEGylated rSak variants to date, do not indicate cardiotoxic hazards associated with the use of these drugs in humans.

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH(2)-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2-to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10-to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine ((125)I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 microg/kg SakSTAR; 50 to 110 microg/kg of core-substitution derivatives with 1 P5; 25 microg/kg for NH(2)-terminal derivatives with 1 P5; 5 to 25 microg/kg with derivatives with 2 P5 or 1 P10; and 7 microg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that mono-PEGylated staphylokinase variants may be used for single intravenous bolus injection.

The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11(+/+)) and MMP-11-deficient (MMP-11(-/-)) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11(-/-) as compared with MMP-11(+/+) mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm(2) at 2 weeks, P<0.001) and those of a 50/50 mixed 129SV/BL6 background (0.030 versus 0.013 mm(2) at 3 weeks, P<0.05). The medial areas were comparable, resulting in intima/media ratios that were significantly increased in MMP-11(-/-) as compared with MMP-11(+/+) arteries, in mice of both the 129SV (1. 0 versus 0.18, P<0.001) and mixed (1.5 versus 0.70, P<0.05) backgrounds. Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in arteries from MMP-11(-/-) mice than in those from MMP-11(+/+) mice (210 versus 48, P<0.001, in pure 129SV mice and 290 versus 150, P<0.01, in mice of the mixed genetic background). Immunocytochemical analysis revealed that alpha-actin-positive and CD45-positive cells were more abundant in intimal sections of MMP-11(-/-) mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11(-/-) mice than in those of MMP-11(+/+) mice (39% versus 6.8% at 3 weeks, P<0. 005). The mechanisms by which MMP-11 could impair elastin degradation and cellular migration in this model remain, however, unknown.

The effects of Enoxaparin with a specific anti-thrombin (anti-IIa) activity of 32 U/mg and a specific anti-factor-XA (anti-Xa) activity of 96 U/mg, and of heparin with a specific anti-IIa and anti-Xa activity of 192 U/mg, on thrombolysis with alteplase (Actilyse) were compared in a randomized blinded study using a combined arterial and venous thrombosis model in the dog. All dogs received an intravenous bolus of 5 mg/kg lysine-acetyl salicylate and 0.5 mg/kg alteplase over 60 min. Twenty-eight dogs were randomly assigned to seven treatment groups: placebo, Enoxaparin 1.5, 3 or 6 mg/kg, or heparin 0.5, 1 or 2 mg/kg, given as a 50% intravenous bolus and a 50% infusion over 2 h. Steady-state plasma levels ranged from 0.37 to 1.0 anti-IIa U/ml and 0.9 to 3.1 anti-Xa U/ml for Enoxaparin and from 0.4 to 2.3 anti-IIa U/ml and 0.42 to 3.2 anti-Xa U/ml for heparin. The activated thromboplastin time with 6 mg/kg Enoxaparin prolonged to 94 +/- 19 s and with 2 mg/kg heparin to > 150 s. The time to reflow was 120 +/- 36 min with placebo, 19 +/- 5 min with 6 mg/kg Enoxaparin (p = 0.03 vs control), and 22 +/- 5 min with 2 mg/kg of heparin (p = 0.03 vs control). Arterial patency, expressed in min reflow during the 180 min observation period correlated significantly with the dose of anticoagulant given (r = 0.73, p = 0.003 for Enoxaparin and r = 0.61, p = 0.012 for heparin).(ABSTRACT TRUNCATED AT 250 WORDS)