Patent Application: US-201314407540-A

Abstract:
a method for the simultaneous measurement of proteolylic enzyme generation and clot strength in plasma or whole blood or any appropriate biological sample derived from blood . the measurement method encompasses the use of a detectable substrate which includes a moiety that can be released upon reaction with the targeted proteolytic enzyme , and elements for measurement of an increase in viscosity of clot strength .

Description:
in the following a typical description of the haemostatic and thrombotic system is given for the present invention with an emphasis on its most important enzyme , thrombin . it is to be noted , however , that the present invention also relates to other enzymes and to other physiological systems , such as other activated proteolytic enzymes ( factors ) of the clotting system in blood and plasma including , plasmin and other activated components of the fibrinolytic system in blood and plasma , and activated complement factors in blood and plasma . to simultaneously measure proteolytic enzyme generation , in particular thrombin generation , and clot strength , the assayed biological sample is applied on a rheometer designed to enable detection of an increase of viscosity due to the formation of fibrin in various shear stress conditions mimicking in vivo physiological conditions . the air - beared rheometer is sensitive enough to detect an increase in viscosity due to the formation of fibrin . the transparent bottom of the sample holder enables transference of fluorescent light emitted by a thrombin sensitive substrate , readily detectable with a built - in fluorometer . the method of the invention , for in vitro simultaneous determination in real time of the course of determined proteolytic enzyme activity and of the strength of the formed fibrin clot in a biological sample , while simulating in vivo blood flow conditions , comprises the following steps : a ) triggering proteolytic enzyme generation in the biological sample so as to enable clotting in said biological sample and contacting said biological sample with a signal substrate causing a detectable signal related to the conversion product released upon reaction of said signal substrate with the proteolytic enzyme generated , b ) displaying the sample mixture obtained in a ) as a thin layer and setting a measuring gap , in particular a zero gap , between the measuring system and the mixture obtained in a ) hold in a vessel suitable for measurement of the detectable signal obtained after reaction of the signal substrate with the proteolytic enzyme , c ) applying a rotational force to the sample mixture obtained in b ), thereby simulating venous or arterial blood flow of a determined in vivo physiological or clinical condition , d ) determining the time course of detectable signal development in the biological sample to provide results of proteolytic enzyme activity over time , especially as a curve , e ) on said triggered biological sample , simultaneously monitoring the increase in viscosity over time with a rotary rheometer , as a measure of the formation of fibrin in the biological sample . the terms “ proteolytic enzyme generation ” and in particular the terms “ thrombin generation ” reflect the appearance and disappearance of the proteolytic enzyme activity , in particular thrombin activity , over time in the triggered biological sample . accordingly the terms “ proteolytic enzyme generation ” and “ proteolytic enzyme activity ” are used interchangeably . the term “ real time ” as used herein is meant to indicate that the course of proteolytic enzyme concentration in the medium is displayed simultaneously with the biological activation and inactivation of the sample that is going on in the reaction vessel . as used herein , the term “ signal substrate ” means a substrate that is cleaved by a proteolytic enzyme generated in the assayed biological sample present in the medium , from which a leaving group is split off which is detectable . in a particular embodiment of the invention , the signal substrate is a fluorogenic substrate , the leaving group of which is detectable by fluorescence detection ,. in particular , the “ signal substrate ” is a substrate that is cleaved by thrombin . suitable fluorogenic signal substrates for thrombin are well known for the skilled person and disclosed especially in ep 0420332 or in wo 03 / 093831 . the expression “ thin layer ” means a layer of assayed sample having a thickness within a range of 0 . 05 to 5 mm and in particular within a range of 1 to 3 mm , for example 2 mm or less . according to the invention , measurement of viscosity as a determination of fibrin formation in the biological sample and determination of proteolytic enzyme generation in said sample are said to be simultaneous because they are performed on a single sample or subsample , e . g ., in one single sample vessel or holder . advantageously , in a particular embodiment of the invention , the measurement of fibrin formation and determination of proteolytic enzyme generation are carried out using a single apparatus , such as a rheometer , in particular a rotary rheometer according to the invention that is specifically designed to also comprise a fluorometer . the assayed sample is a biological sample usually consisting of blood i . e ., whole blood , or a product derived from blood such as plasma in particular platelet - rich plasma ( prp ), platelet - poor plasma ( ppp ) or platelet - free plasma ( pfp ). blood is usually collected in tubes that contain either sodium citrate or edta , or the like , so that free calcium ions in the blood are bound and proteolytic enzyme formation , in particular plasmin or thrombin formation and clotting is prevented during sample collection and storage . the invention provides the possibility of using biological samples , especially blood samples in a range as small as 20 to 60 μl and up to approximately 1 ml . according to the invention triggering proteolytic enzyme generation is carried out in a known manner , with activation factors . activation factors , also designated protease activators for carrying out the present method include phospholipids , tissue factor , in particular soluble tissue factor , or recombinant human tissue factor , thromboplastin , kaolin , and elagic acid . depending on the biological sample used , they may be added in the first subsample to trigger proteolytic enzyme activity . they are also advantageously added to the mixture eliciting a constant known proteolytic enzyme activity in particular plasmin or thrombin activity . the activation factors may be used with further components necessary or advantageous to start the coagulation reactions such as calcium ions . indeed , in order to start reactions eliciting proteolytic enzyme generation , in particular plasmin or thrombin generation , calcium should be added shortly before the start of the measurement . in an embodiment of the invention , as illustrated in the examples calcium is added as fluca , i . e ., as a mixture of fluorogenic substrate and cacl2 . this fluca mixture may be prepared as disclosed in the examples . however , in case blood is not collected in sodium citrate , or the like , this addition of calcium may not be necessary and the fluorogenic substrate is thus added as a solution . this may be the case when the method is used in a way that makes it possible to start the experiment within minutes after blood taking . in a particular embodiment of the invention , the method provides in vitro simultaneous determination in real time of the course of thrombin activity and of the strength of the formed fibrin clot . when the proteolytic enzyme is thrombin , thrombin activity against time may be presented as a curve of thrombogram ™ ( tg ) which is characterized by an initiation phase providing minute amounts of thrombin followed by a burst of activity . blood forms a clot at the very beginning of the burst and almost all thrombin is formed after the clot has formed . the thrombogram ™ thus shows various phases including lag time , propagation phase , time to peak and peak height and etp . the term “ lag time ” as used herein is meant to indicate the time ( min ) it takes before thrombin formation starts . as used herein , the term “ peak height ” ( nm ) means the maximal thrombin activity attained during the assay on a tested sample . the term “ time to peak ” ( min ), as used herein , means the time between the start of the reaction and the moment that the peak is reached . the term “ etp ” ( endogenous thrombin potential ), as used herein , means the time integral of the curve until the moment of ( near ) zero thrombin activity is attained , corresponding to the area under the curve ( nm . min ). the signal substrate which is used in the present method is preferably selected from the group of compounds comprising a leaving group , said leaving group providing a detectable conversion product upon reaction of said compound with the proteolytic enzyme formed . this conversion product is determined by spectroscopy . in a particular embodiment the signal substrate is a fluorogenic substrate , and its leaving group emits fluorescence . accordingly , in a particular embodiment of the invention , said leaving group is a fluorescent group . in a particular embodiment of the invention for thrombin activity measurement , the leaving group , especially the fluorescent group is present on a substrate which is sensible to thrombin and undergoes a splitting reaction of the leaving group . in a particular embodiment , the signal substrate is a fluorogenic substrate selectively hydrolyzed by thrombin , having a moderate binding affinity for thrombin and a low kinetic constant and in particular is a synthetic substrate for thrombin , coupled with a fluorescent molecule . in a particular embodiment of the invention , the fluorogenic substrate is an oligopeptide having a sequence of 2 to 30 amino acids residues coupled with a fluorescent molecule . as an example , the oligopeptide has a terminal lysine or arginine for coupling with a fluorescent molecule . a suitable and preferred signal substrate for carrying out the method of the present invention using a fluorescence detector , on whole blood or plasma is ( z - gly - gly - arg ) 2 rho110 , a rhodamine - based thrombin substrate . rhodamine is extremely useful given its high quantum yield , excitation and emission wavelengths that have less overlap with the absorption spectrum of hemoglobin , and since it is not significantly consumed during the time of the assay . alternatively , for plasma measurements z - gly - gly - arg - amc ( backen switzerland ) can be used as a thrombin substrate , which releases amc ( 7 - amino - 4 - methylcoumarin ) and is measured by a 390 nm excitation and a 460 nm emission filter set . when the proteolytic enzyme generation measured is the generation of plasmin , the substrate may be bis -( cbz - l - phenylalanyl - l - arginine amide ), dihydrochloride coupled to rhodamine 110 . another fluorogenic substrate suitable for the detection of plasmin generation is h - ala - leu - lys - amc ( stago , france ). it has been reported in the prior art that in the particular case of measurement of thrombin generation , thrombin and its substrate do not react linearly with each other . in order to compensate for non - linearity of the reaction rate of thrombin with its substrate , depending upon thrombin concentration in the biological sample , measurement of a mixture eliciting a constant known thrombin activity is performed in parallel on a non clotting second part of the assayed sample ( second subsample ). this step provides calibration for proteolytic enzyme activity measurement in the first part of the sample . calibration is thus advantageously performed when the measured proteolytic enzyme activity is thrombin activity . rather plasmin generation measurement can be performed without a calibration step . accordingly , in a particular embodiment , for the purpose of carrying out the invention described herein , including a calibration of the proteolytic enzyme activity , the method comprises : an initial step of dividing the biological sample in at least two parts , designated subsamples , including a first subsample and a second subsample , performing steps a ) to e ) of the method for the simultaneous detection of determined proteolytic enzyme generation and fibrin formation of the invention are performed on the first subsample and , performing the following steps on the second subsample for calibration of the results of measurement of proteolytic enzyme activity : f ) adding to the non - clotting second subsample , a calibrator compound harbouring a constant stable activity on the signal substrate for said determined proteolytic enzyme said calibrator compound being otherwise inert in the coagulation reactions of the subsample ( being inert to physiological antithrombin agents in blood ), and contacting the obtained mixture of said calibrator compound and second subsample with a signal substrate causing a detectable signal related to the conversion product released upon reaction of said signal substrate with the proteolytic enzyme of said second subsample , g ) submitting said mixture obtained in step f ) with the second subsample to steps b ) and c ) of the method of the invention as defined herein , h ) determining the time course of detectable signal development in the second subsample to provide results of proteolytic enzyme activity over time especially as a curve ; i ) comparing the results obtained in step g ) to the results of proteolytic enzyme activity obtained in step d ) to derive calibrated results of proteolytic enzyme activity in time in the first subsample , in particular as a calibrated curve . suitable calibrator compound with a constant known stable proteolytic activity for carrying out the method of the present invention , as defined above , include α 2 m - thrombin complex ( α2 - m - t ), staphylocoagulase - prothrombin complex , and gamma thrombin . alternatively , any proteolytic enzyme can be used which is modified in its secondary recognition sites in that its active center for proteolysis remains intact but its functional interaction with proteins in the reaction mixture is abolished , so that it is regarded as inert in the coagulation reactions taking place in the reaction mixture . the methods and reagents of the invention are suitable for use in measuring properties of blood to form fibrin clot and properties of blood with respect to proteolytic enzyme generation . a rheometer according to an embodiment of the invention , designed to carry out the measurement of proteolytic enzyme generation and the simultaneous measurement of clot strength is shown in fig2 and 3 . said rheometer comprises a head part 1 having a motor 2 driving a shaft 3 supporting a cone element 4 at its lower end . the tip 5 of the cone element 4 is oriented downward and is facing an upper plate 6 of a base part 7 . the angle θ of the cone 4 is for example between 0 , 1 ° and 5 °, and is preferably 0 , 5 °. the head part 1 also comprises means 8 for measuring the rotational speed of the shaft 3 and / or the torque exerted by the motor 2 to the shaft 3 . optionally , a dark hood 9 is slidably mounted on the shaft 3 . in this case , the position of the hood 9 can be adjusted by corresponding means 10 . the position of the head part 1 relative to the base part 7 is controlled by means 11 , in order to set the gap g ( measuring gap ) between the tip 5 of the cone 4 and the upper plate 6 of the base part 7 , or between the tip 5 of the cone 4 and the bottom surface 14 ′ of a holder 14 . a fluorometer 12 is mounted into the base part 7 for quantifying the fluorescence signal emitted by a sample 13 placed on the upper plate 6 . the sample 13 is for example inserted into a cup - like holder 14 . both the holder 14 and the upper plate 6 are transparent , at least partially , to enable the quantification of the fluorescence signal emitted by the sample 13 . during an assay , the sample holder 14 is placed on the upper plate 6 of the base part 7 and the cone 4 is lowered by the means 11 , to adjust the gap g . said gap is for example between 10 μm and 2 mm , preferably not more than 100 μm . the hood 9 is also lowered to cover the sample holder 14 so as to avoid any loss of fluorescence signal . during the assay , the fluorescent signal emitted by the sample 13 is also measured by the fluorometer 12 , through the holder 14 and the upper plate 6 , in order to determine changes in thrombin concentration over time . according to the invention , when using the rheometer , the rotational force applied to the biological sample is selected to reflect the blood in said vascular setting . hence , for arterial setting the shear rate is in the range of 800 to 3000 s - 1 ; in a venous setting the rotational force is in the range of 50 to 800 s - 1 . when an oscillatory force is applied it may be in a range of 0 . 1 s - 1 to 300 s - 1 . the method of the invention is performed applying a rotational force to the mixture obtained with the biological sample in step a ). by rotating the measuring system , the shear rate of venous and arterial blood flow occurring in vivo can be simulated , enabling to study in vitro blood coagulation / clot lysis under flow , rendering information on platelet function and fibrinolysis under conditions that more closely resemble the in vivo situation . accordingly , the invention provides reagents and methods to study hemostasis in a physiological setting to determine coagulation propensity or in a pathological setting , especially to determine or to monitor a clinical condition in a patient , or to perform therapy planning or therapy monitoring . in a particular embodiment , the methods , reagents and kits of the invention are used for the determination of a risk to face thrombosis or bleeding in a patient . the invention , in providing operating conditions mimicking in vivo blood flow may be used to study various vascular settings , in particular arterial , venous or tumor microcirculation . according to a particular aspect of the present invention said first subsample of the assayed biological sample further comprises a drug or a pharmaceutical agent to be tested , for its influence on the proteolytic system under study , such as the haemostatic - thrombotic system . suitable pharmaceutical agents which can be tested in the present method are antithrombotic agents , such as anti - platelet agents and anticoagulating agents , for example heparin , dermatan sulphate , a direct thrombin - inhibitor , for example hirudin , argatroban or melagatran , and a factor xa inhibitor , for example thick anticoagulant protein . according to another aspect of the invention , the method is performed in order to monitor the effects of drugs administered to a patient on the haemostatic - thrombotic system . such drugs include anticoagulant drugs , especially all types of heparins , direct thrombin inhibitors or other antithrombodic agents such as those cited above . by measuring the fluorescence in the thin layer of biological sample formed between the plate containing the assayed biological sample and the measuring system , the effect of the inner filter effect described above is reduced . the invention also relates to a method as defined herein when used : for detecting or monitoring the effect of determined substances , especially a drug or a pharmaceutical agent on thrombin activity and clot strength in a biological sample , wherein said determined substance ( s ) is ( are ) added to the sample to be assayed or is ( are ) added during thrombin generation or for monitoring effects of drugs on a biological sample of a patient under treatment with this drug or for screening substances to determine their interacting capacity with thrombin generation or fibrinolysis or coagulation , to assess deficiencies in molecules in the haemostatic - thrombotic system or , to determine the minimal amount of thrombin necessary to initiate clot initiation . the invention also relates to a kit for the determination of the course of proteolytic enzyme activity and for the simultaneous determination of the strength of the formed fibrin clot in a biological sample , comprising : in a disposable sample holder having a transparent bottom surface , suitable for use on a rotary rheometer , a composition of a signal substrate causing a detectable signal related to the conversion product released upon reaction of said signal substrate with the proteolytic enzyme generated in the triggered sample and a composition for triggering the proteolytic enzyme activity in the sample , optionally a calibrator compound compound harbouring a constant stable proteolytic enzyme activity on the signal substrate , optionally a suitable buffer solution for addition to the biological sample , such as bsa5 buffer comprising 20 mm hepes and bovin serum albumin 5 mg / ml , at ph = 7 . 35 . the invention will now be further illustrated by the following examples which are not to be construed as limiting the scope of the invention in any respect . blood was obtained through venipuncture ( 1 volume tri - sodium citrate 0 . 13m to 9 volumes blood ). free flow or minimal suction should be employed , and vacuum containers avoided . platelet poor plasma ( ppp ) was obtained by centrifugation of blood twice at 2630 g for 10 min . recombinant relipidated tissue factor ( rtf ) not containing polybrene or ca ++ was from dade behring ( marburg , germany ). for each experiment , a fresh mixture of tissue factor ( tf ) and phospholipids vesicles ( pl ) ( avanti , alabaster , ala ., usa ) was prepared , resulting in a final concentration of 5 pm tf and 4 μm pl in hepes buffer ( 20 mm , ph 7 . 35 ) containing 5 mg / l bsa ( sigma , a - 7030 ). recombinant tissue plasminogen activator ( tpa ) was obtained from boehringer ingelheim gmbh , germany as actylase ™. fluorogenic substrate , ( z - gly - gly - arg ) 2 rho110 , was obtained from diagnostica stago ( asnieres , france ) and used at a concentration of 300 μm . upon splitting by thrombin , it releases the fluorescent rhodamine ( rho ), which is measured by a 470 nm excitation and a 520 emission filter set . the fluorogenic plasmin substrate , h - ala - leu - lys - amc , was obtained from diagnostica stago and used at a final concentration of 200 μm . the by plasmin released fluorescent 7 - amino - 4 - methylcoumarin ( amc ) is measured by a 365 nm excitation and a 430 nm emission filter set . a fresh mixture of fluorogenic substrate and cacl 2 ( fluca ) was prepared at 37 ° c . for each experiment , resulting in a final concentration of 300 μm rho and 16 . 7 mm cacl 2 in hepes buffer ( 20 mm , ph 7 . 35 ) containing 60 g / l bsa . the calibrator , α2m - thrombin complex ( α2mt ), was prepared in house as previously described [ 2 ]. the concentration of α2m - thrombin is measured by its ability to hydrolyze the chromogenic substrate s2238 . this amidolytic activity of the preparation was adjusted to have in the final reaction the same activity as 100 nm human thrombin . after adding bovine antithrombin and heparin ( leo ) this material was ready for use as a thrombin calibrator . using a signal substrate with a fluorescent leaving group , thrombin generation was measured via a fluorescence detector . the strength of the formed fibrin clot was monitored by an oscillating or rotating measuring system of a specially designed rheometer ( physica mcr 301 , anton paar germany gmbh , ostfildern ). a total of 700 μl ( in case of plasma ) or 800 μl ( in case of blood ) sample and substrate was added on the thermostated sample holder at 37 ° c . of the rheometer . this mixture contained ( and was prepared by adding in the following order ) sample ( 468 μl plasma or 700 μl blood ); tf - pl mix ( or calibrator ) ( 116 μl in case of plasma ; 50 μl in case of blood ) and fluca ( 116 μl in case of plasma ; 50 μl in case of blood ). immediately after addition of the fluca , the viscosity ( or torque in case of oscillation of the measuring system ) and fluorescence were measured every 10 seconds and every second at 470 / 520 nm respectively . thrombin generation in relation to clot strength based on viscosity detection thrombin generation was triggered in whole blood as indicated above on the glass of an mcr301 rheometer with a built - in fluorometer . immediately after addition of the fluca solution , the measuring system of the rheometer was set to a zero gap position and brought into oscillation , at a frequency of 10 hz and an angle of 1 °. torque and fluorescence were measured as indicated above , both for the triggered sample ( first part of the sample , i . e ., first subsample ) and the sample containing the calibrator ( second part of the sample , i . e ., second subsample ). the use of the calibrator avoids the donor - dependent influence of the fluorescent signal through absorption of light and quenching of the fluorescence . based on the calibrator curve and after correction for the α2m - thrombin present in the sample which is still able to convert the substrate , the thrombin generation curve for the triggered sample was determined , as described above . the mean of two measurements is shown in fig3 ( upper panel ). clot initiation ( shown as torque in the lower panel ) already started at the very beginning of the thrombin generation . interestingly , by adding tissue plasminogen activator ( tpa ) at a concentration of 300 u / ml , fibrinolysis could be followed in the blood sample ( inset ). thrombin generation in relation to fibrin formation based on viscosity detection ; simulation of blood flow thrombin generation was triggered in platelet - poor plasma or whole blood as indicated above on the glass of an mcr301 rheometer with a built - in fluorometer . immediately after addition of the fluca solution , the measuring system of the rheometer was set to a zero gap position and brought into rotation . this rotation of the measuring system allowed simulation of the blood flow , a shear rate of 100 - 300 / s corresponding to venous blood flow and above 800 / s to arterial blood flow . viscosity and fluorescence were measured as indicated above , both for the triggered sample and the sample containing the calibrator . measurements were done in triplicate and the mean is shown in fig5 a ( platelet - poor plasma ) and 5 b ( whole blood ). in platelet - poor plasma , shear rates higher than 100 / s resulted in decreased thrombin generation and clot strength . the effect on clot strength was repeated in whole blood , however the effect on thrombin generation was different . visualization of the effect of doolittle peptide on thrombin generation and clot strength thrombin generation was triggered in platelet - poor plasma as indicated above on the glass of an mcr301 rheometer with a built - in fluorometer . increasing concentrations of doolittle peptide ( gly - pro - arg - pro ), 0 . 5 mm up to 2 mm , were added to the sample and corresponding calibrator . doolittle peptide binds with a high affinity to fibrinogen , resulting in the inhibition of fibrin polymerization . immediately after addition of the fluca solution , the measuring system of the rheometer was set to a zero gap position and brought into rotation , resulting in a shear rate of 300 / s . viscosity and fluorescence were measured as indicated above both for the triggered sample and the sample containing the calibrator . measurements were done in duplicate and the mean is shown in fig6 . as expected , increasing amounts of doolittle peptide significantly decreased the viscosity of the sample compared to the control sample ( upper panel ). the effect on the thrombin generation was minimal ( lower panel ). thrombin generation was triggered in control and defibrinylated plasma as indicated above on the glass of an mcr301 rheometer with a built - in fluorometer . immediately after addition of the fluca solution , the measuring system of the rheometer was set to a zero gap position and brought into rotation , resulting in a shear rate of 300 / s . viscosity and fluorescence were measured as indicated above . results are shown in fig7 . as expected , defibrinylation of plasma resulted in the complete abolishment of the increase in viscosity ( upper panel ) without a clear effect on the thrombin generation ( depicted as etp ; lower panel ). plasmin generation and clot strength in tissue plasminogen activator - triggered platelet - poor plasma clot initiation was triggered in platelet - poor plasma as indicated above in the presence of 100 u / ml tpa on the glass of an mcr301 rheometer with a built - in fluorometer . immediately after addition of the plasmin substrate solution containing ca , the measuring system of the rheometer was set to the measuring gap position and brought into rotation ( 300 / s ). fluorescence and viscosity were measured as indicated above , both for the triggered sample and the sample containing the calibrator . raw fluorescence and viscosity data are shown in the upper and lower panel of fig8 , respectively . results clearly indicate activation of plasmin by tpa , resulting in the degradation of fibrin ( decrease in viscosity ). the present invention accordingly offers a convenient test method to determine simultaneously and in real time the course of a proteolytic activity , in particular thrombin activity , and the strength of the clot in a blood / plasma sample . the test provides a continuous signal thereby giving more valuable and accurate information regarding such parameters as lag time , peak height and time to peak as compared with methods that only use end - point determinations of the amount of product formed . importantly , the present invention allows the in vitro investigation of thrombin generation and clot strength in conditions resembling in vivo shear rates in venous and arterial blood flow , enabling valuable information on platelet function and fibrinolysis . simulating shear rates of venous and arterial blood flow is important given the fact that a clot must resist shear stress at the site of the injury . 1 konings j , govers - riemslag j w , philippou h , mutch n j , borissoff j i , allan p , mohan s , tans g , ten cate h , ariens r a . factor xiia regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin . blood . 2011 ; 118 : 3942 - 51 . 2 hemker h c , giesen p , al dieri r , regnault v , de smedt e , wagenvoord r , lecompte t , beguin s . calibrated automated thrombin generation measurement in clotting plasma . pathophysiol haemost thromb . 2003 ; 33 : 4 - 15 . 3 beguin s , kumar r . thrombin , fibrin and platelets : a resonance loop in which von willebrand factor is a necessary link . thromb haemost . 1997 ; 78 : 590 - 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