The adsorption of dabigatran is as efficient as addition of idarucizumab to neutralize the drug in routine coagulation assays.

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.

Measurement of B-domain-deleted ReFacto AF activity with a product-specific standard is affected by choice of reagent and patient-specific factors.

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.

The amplitude of coagulation curves from thrombin time tests allows dysfibrinogenemia caused by the common mutation FGG-Arg301 to be distinguished from hypofibrinogenemia.

INTRODUCTION: Thrombin time (TT) tests are useful for diagnosing coagulation disorders involving abnormal fibrinogen but do not allow us to distinguish between qualitative and quantitative defects. However, with the widening availability of optical coagulation automates, more information about the coagulation process is becoming increasingly accessible. METHODS: In this study, we compared the coagulation curves of TT tests carried out with plasma from healthy donors with those from patients with acquired low Clauss fibrinogen levels or with dysfibrinogenemia caused by a heterozygous point mutation in the fibrinogen γ-chain that results in a p.Arg301(275)Cys substitution. The functional fibrinogen levels of these three groups of samples were also measured with the Clauss method, and their fibrinogen protein levels were determined by ELISA. RESULTS: Our data indicate that the amplitude and maximal velocity of coagulation curves from plasma samples from FGG p.Arg301(275)Cys dysfibrinogenemic patients were comparable to those from plasma samples with fibrinogen in the normal range, whereas the amplitude of coagulation curves from patients with acquired low fibrinogen levels was lower. CONCLUSIONS: Examination of the amplitude of coagulation curves generated during TT tests may provide additional information to enable the differential diagnoses of diseases following a low fibrinogen measurement by the Clauss method.

The addition of idarucizumab to plasma samples containing dabigatran allows the use of routine coagulation assays for the diagnosis of hemostasis disorders.

BACKGROUND: The anticoagulant effect of dabigatran can be approximated by its prolongation of routine coagulation assays. Consequently, dabigatran also interferes with thrombophilia screening or with diagnosing hemostasis disorders that have developed after the initiation of anticoagulant treatment, such as vitamin K deficiency or acquired hemophilia A. OBJECTIVES: This study was carried out to determine whether idarucizumab, a humanized antibody fragment that binds dabigatran, could fully neutralize dabigatran in routine diagnostic coagulation assays conducted in vitro, thereby preventing false-positive or false-negative diagnostic readouts. METHODS: Preliminary experiments identified coagulation assays that were sensitive to dabigatran, and identified a concentration of idarucizumab that neutralized the effects of dabigatran. These assays were then carried out with patient and control plasma samples spiked with dabigatran, with or without a molar excess of idarucizumab. RESULTS: Dabigatran altered the prothrombin time, activated partial thromboplastin time and thrombin time, and the measurement of intrinsic and extrinsic factor levels. Screening and confirmation tests used for lupus anticoagulant detection were prolonged by dabigatran, falsely suggesting the presence of lupus anticoagulant. Conversely, the addition of dabigatran falsely corrected an abnormal activated protein C resistance ratio. Addition of idarucizumab completely normalized these measurements, and allowed the correct identification of normal and abnormal samples with these assays. CONCLUSIONS: In vitro addition of idarucizumab to plasma samples containing dabigatran fully neutralizes the drug, and facilitates the use of routine coagulation assays to allow the diagnosis of hemostasis disorders that may be concurrently present in patients taking dabigatran.

Toxicology studies with recombinant staphylokinase and with SY 161-P5, a polyethylene glycol-derivatized cysteine-substitution mutant.

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.

Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase.

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.

Accelerated neointima formation after vascular injury in mice with stromelysin-3 (MMP-11) gene inactivation.

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.

Comparative effects of tissue plasminogen activator, streptokinase, and staphylokinase on cerebral ischemic infarction and pulmonary clot lysis in hamster models.

BACKGROUND: The effects of alteplase (rtPA), streptokinase, and staphylokinase (rSak) on focal cerebral ischemia (FCI) and on pulmonary clot lysis (PCL) were studied in hamsters. METHODS AND RESULTS: ++FCI was produced by ligation of the left middle cerebral artery (MCA) and common carotid artery (CCA) and a 10-minute occlusion of the right CCA. FCI was measured after 24 hours by 2,3, 5-triphenyltetrazolium chloride staining. (125)I-fibrin-labeled plasma clots were injected via the jugular vein, and clot lysis was determined from residual radioactivity at 90 minutes. Study drugs were given intravenously over 60 minutes. FCI increased from 1.2 (0. 27 to 2.3) mm(3) (median and 17th to 83rd percentile range, n=24) in controls to 19 to 27 mm(3) with thrombolytic agent, with maximal rates at 0.13+/-0.05 mg/kg rtPA, 0.23+/-0.09 mg/kg streptokinase, and 0.037+/-0.025 mg/kg rSak. PCL increased from 18+/-2% (mean+/-SEM, n=27) in controls to approximately 85% with thrombolytics, with maximal rates at 0.12+/-0.03 mg/kg rtPA, 0.17+/-0.05 mg/kg streptokinase, and 0.018+/-0.002 mg/kg rSak. All agents caused maximal FCI and PCL rates at similar doses without alpha(2)-antiplasmin and fibrinogen depletion. Injection of 6 mg/kg human plasminogen combined with streptokinase caused a "systemic fibrinolytic state" with fibrinogen depletion. Maximal rates of FCI were obtained with 0.097+/-0.077 mg/kg streptokinase (P=0.26 versus streptokinase alone) and of PCL with 0.010+/-0.002 mg/kg (P=0.006 versus streptokinase alone). CONCLUSIONS: Thrombolytic agents cause similar dose-related extension of FCI after MCA ligation and PCL, irrespective of the agent or systemic plasmin generation.

Comparative effects of enoxaparin and heparin on arterial and venous clot lysis with alteplase in dogs.

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)

Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator.

rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative fibrinolytic properties of staphylokinase and streptokinase in animal models of venous thrombosis.

The thrombolytic and pharmacokinetic properties of staphylokinase were compared with those of streptokinase in hamsters with a pulmonary embolus produced from human plasma or from hamster plasma, and in rabbits with a jugular vein blood clot produced from rabbit blood. In both models, a continuous intravenous infusion of staphylokinase and streptokinase over 60 min in hamsters or over 4 h in rabbits, induced dose-dependent progressive clot lysis in the absence of significant systemic activation of the fibrinolytic system. The results of thrombolytic potency (clot lysis at 30 min after the end of the infusion, in percent, versus dose administered, in mg/kg) were fitted with an exponentially transformed sigmoidal function (formula: see text) and the maximal percent clot lysis (c), the maximal rate of lysis (z = 1/4 ac.eb) and the dose at which the maximal rate of lysis is achieved (b) were determined. In hamsters with a pulmonary embolus produced from human plasma, streptokinase had a somewhat higher thrombolytic potency than staphylokinase, as revealed by a higher z value (2,100 +/- 1,100% lysis per mg/kg streptokinase administered versus 1,100 +/- 330% lysis per mg/kg for staphylokinase). In hamsters with a pulmonary embolus produced from hamster plasma, staphylokinase had a somewhat higher thrombolytic potency than streptokinase (z = 1,600 +/- 440 versus 1,200 +/- 370% lysis per mg/kg). Staphylokinase had a higher thrombolytic potency than streptokinase in rabbits,as revealed by a higher z-value (950 +/- 350% lysis per mg/kg staphylokinase administered versus 330 +/- 39% lysis per mg/kg for streptokinase) and a lower b-value (0.035 +/- 0.010 mg/kg staphylokinase versus 0.091 +/- 0.008 mg/kg for streptokinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model.

A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 microliters 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. Recombinant t-PA (home-made rt-PA or Activase) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in microgram/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1 + e-a(ax-eh] and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = 1/4 ac.eb) were determined. With rt-PA, these parameters were c = 72 +/- 6% (mean +/- SEM), b = 0.19 +/- 0.08 mg/kg, z = 68 +/- 25% lysis per mg/kg, with corresponding values of 87 +/- 5%, 0.07 +/- 0.03 mg/kg and 150 +/- 38% lysis per mg/kg for Activase (p = NS). Deletion of the finger and growth factor domains in rt-PA (rt-PA-delta FE) was not associated with marked alteration of the thrombolytic potency (c = 90 +/- 30%, b = 0.34 +/- 0.35 mg/kg, and z = 54 +/- 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)