scholarly journals Comparison of platelet-derived and plasma factor VIII efficacy using a novel native whole blood thrombin generation assay

2015 ◽  
Vol 13 (12) ◽  
pp. 2210-2219 ◽  
Author(s):  
C. K. Baumgartner ◽  
G. Zhang ◽  
E. L. Kuether ◽  
H. Weiler ◽  
Q. Shi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Thrombin Generation Assay ◽  
Plasma Factor

scholarly journals Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

2008 ◽  
Vol 6 (3) ◽  
pp. 486-493 ◽  
Author(s):  
M. TROSSAËRT ◽  
V. REGNAULT ◽  
M. SIGAUD ◽  
P. BOISSEAU ◽  
E. FRESSINAUD ◽  
...  
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Thrombin Generation Assay

Binding To Phospholipid Protects Factor VIII From Inhibition By Human Antibodies

1981 ◽  
Author(s):  
T W Barrowcliffe ◽  
E Gray ◽  
G Kemball-Cook
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Total Phospholipid ◽  
Fluid Phase ◽  
Clinical Applications ◽  
Factor Ix ◽  
Two Stage ◽  
Human Antibodies ◽  
Thrombin Generation Assay ◽  
Detectable Antigen

Previous studies with activated Factor IX concentrates have suggested that they may contain a form of Factor VIII clotting activity (VIII:C) which is partly protected from inactivation by antibodies. A possible mechanism for such protection is binding to phospholipid. The interaction between Factor VIII, phospholipid and human antibodies to Factor VIII was studied by a two-stage clotting assay, and by a fluid-phase immunoradiometric assay for Factor VIII clotting antigen (VIII C:Ag).In the two-stage thrombin generation assay, Factor VIII:C was rapidly destroyed by human antibodies, even in the presence of optimal phospholipid. However, preincubation of Factor VIII with phospholipid before addition of antibody protected the Factor VIII from inactivation, resulting in the production of much more thrombin.In assays of VIII C:Ag, pre-incubation of Factor VIII with phospholipid before addition of labelled antibody reduced the amount of detectable antigen. The reduction was greater with increasing phospholipid concentration, up to 60% of the original antigen being ‘lost’ at a total phospholipid concentration of around 250 μg/i.u.These results suggest that human antibodies to Factor VIII are directed largely at its phospholipid binding site. The protection of Factor VIII from inactivation by complexing with phospholipid could have important clinical applications in treatment of haemophiliacs with inhibitors.

Native Whole Blood Thrombin Generation Assay Evaluates Therapeutic Efficacy Of Plasma and Platelet-Derived FVIII

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1093-1093
Author(s):  
Christina K Baumgartner ◽  
Qizhen Shi ◽  
Robert R. Montgomery
Keyword(s):  
Gene Therapy ◽  
Replacement Therapy ◽  
Whole Blood ◽  
Therapeutic Efficacy ◽  
Thrombin Generation ◽  
Generation Rate ◽  
Gene Therapy Approach ◽  
Thrombin Generation Assay ◽  
Recombinant Fviii ◽  
Dose Dependent

Abstract Factor VIII (FVIII) gene therapy is a promising approach to potentially permanently and cost-effectively correct the bleeding phenotype of hemophilia A patients and improve patients quality of life. Our group has developed a successful gene therapy approach in which FVIII expression is targeted to platelets. Platelet expressed FVIII protects hemophilic mice from lethal blood loss after vessel injury. Most importantly this therapy does not induce FVIII inhibitory antibodies and is even successful in the treatment of mice with pre-existing high titer inhibitors. Therefore this approach is among the first to hold promise for patients who develop inhibitory antibodies against FVIII that render FVIII replacement therapy ineffective. Levels of platelet expressed FVIII achieved by gene therapy may vary between individuals due to differences in ex vivotransduction and gene expression efficiency. We determined hemostatic efficacy over a wide therapeutic dose range with a novel native whole blood thrombin generation assay. Tracking the correction of abnormal bleeding phenotypes during the treatment of patients with hemostatic disorders is crucial to evaluate success of therapy. Global coagulation assays in contrast to single clotting factor assays are desirable to better understand the overall hemostatic condition of patients. Here we evaluated thrombin generation using a modified protocol of a recently described whole blood assay. In our native assay we initiated coagulation without the addition of tissue factor. Sole recalcification of whole blood resulted in thrombin generation with high reproducibility. Lag time (LT) determined in blood from C57BL/6 WT mice was 6 ± 0.2 min (Mean ± SEM) , thrombin generation rate was 58 ± 6 nM/min and thrombin peak was 188 ± 7 nM. In contrast, FVIII deficient blood had negligible thrombin generation with 39 ± 7 min LT, 1.4 ± 0.3 nM/min thrombin generation rate and 12 ± 3 nM thrombin peak. Spiking hemophilic blood with increasing concentrations of recombinant FVIII ex vivo resulted in a dose dependent increase in thrombin generation. Reconstitution of hemophilic blood with FVIII to a 1%, 10% and 100% level shortened LT to 19 ± 1, 12 ± 0.3 and 9 ± 0.5 min, respectively. To evaluate efficacy of platelet-derived FVIII we utilized a newly developed transgenic mouse model that expresses high levels of FVIII in platelets. Homozygous mice express platelet FVIII levels corresponding to 20% endogenous FVIII in whole blood. We combined different ratios of FVIII deficient blood with blood from platelet FVIII expressing transgenic mice. At low ratios of transgenic blood, similar to ex vivospiking with recombinant FVIII, thrombin generation parameters were dose-dependent. Remarkably, a corresponding dose of as low as 0.2% platelet-derived FVIII significantly elevated thrombin generation above FVIII deficient blood and had comparable therapeutic efficacy as a 5-fold higher dose of recombinant FVIII (LT, 18 ± 2 vs 19 ± 1). Similarly, efficacy of 1.5% of platelet-derived FVIII compared with the 6.7-fold higher, 10% dose of recombinant FVIII (LT, 13 ± 1 vs 12 ± 0.3). Further increase of thrombin generation was noticed with platelet FVIII expressing transgenic blood ratios corresponding to 2% and 5% FVIII levels (LT, 11 ± 0.3 and 8.7 ± 0.3 min, respectively). Interestingly, our native assay showed that the platelet FVIII expressing transgenic blood ratio corresponding to a FVIII level of only 5% was sufficient to induce maximal thrombin generation, similar to that obtained with undiluted transgenic blood (LT, 8.7 ± 0.6 min). A similar FVIII dose-dependency was identified for additional thrombin generation parameters including endogenous thrombin potential, thrombin peak, peak time and thrombin generation rate. We conclude that this native whole blood thrombin generation assay could be used to track therapeutic efficacy of hemophilia A treatment. Using this assay, our data indicate that similar to FVIII replacement therapy our previously established platelet targeted FVIII gene therapy approach enhances hemostasis over a wide therapeutic dose level. This is of great importance because levels of platelet expressed FVIII achieved upon gene therapy in mice vary. In agreement with our previous reports our data from native whole blood thrombin generation assay confirm that at lower FVIII dose levels platelet targeted FVIII gene therapy might be more efficient than factor replacement therapy. Disclosures: No relevant conflicts of interest to declare.

Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3798-3798
Author(s):  
Lilley Leong ◽  
Irina N. Chernysh ◽  
Yifan Xu ◽  
Cornell Mallari ◽  
Billy Wong ◽  
...  
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Neutralizing Antibodies ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Clot Formation ◽  
Factor Vii ◽  
Wild Type ◽  
Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

scholarly journals Thrombin generation assay using factor IXa as a trigger to quantify accurately factor VIII levels in haemophilia A

2011 ◽  
Vol 9 (8) ◽  
pp. 1549-1555 ◽  
Author(s):  
M. NINIVAGGI ◽  
Y. DARGAUD ◽  
R. Van OERLE ◽  
B. De LAAT ◽  
H. C. HEMKER ◽  
...  
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Haemophilia A ◽  
Thrombin Generation Assay ◽  
Factor Ixa

scholarly journals Thrombelastography (TEG) and Thrombin Generation Assay (TGA) in Severe Hemophilia Following Factor Replacement Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4666-4666 ◽  
Author(s):  
Tania T. Sarker ◽  
Donald Brophy ◽  
Meera B. Chitlur
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Thrombin Generation ◽  
Factor Activity ◽  
Treatment Frequency ◽  
Thrombin Generation Assay ◽  
Fviii Activity ◽  
The Mean ◽  
Life Factor ◽  
Time Point

Abstract Background: Monitoring therapy in hemophilia is a major challenge. Measurement of factor levels is time consuming and not available in time to make clinical decisions. With the introduction of extended half-life factor products, determination of treatment frequency becomes important. Global hemostatic assays such as Thrombelastography (TEG) and Thrombin Generation Assay (TGA) may improve monitoring. Focused toward individualizing therapy, these assays may help determine treatment frequency based not just on Factor VIII PK (pharmacokinetic), but also on total hemostatic potential. Objective: To determine the correlation between TGA and TEG parameters, and Factor activity and half-life (t1/2). Design/Methods: With IRB approval and participant consent baseline FVIII activity was obtained at enrollment, 15minutes, 1, 4, 8, 24 and 48 hours post factor replacement in patients who had not received replacement factor for a minimum of 72 hours and were not bleeding. FVIII:C, TEG, and TGA at each time point were measured. Non-compartmental PK analysis was performed on each individual patient profile to measure Factor VIII terminal half-life (t 1/2), mean normalized factor clearance rate and volume of distribution at steady-state (Vdss). Pearson correlation statistical analyses on other variables were performed using JMP ¨ Pro version 12.0.1 (SAS Institute, Cary, NC, USA) Results: 27 patients with hemophilia have enrolled, with a median age of 14 years (range: 2-24 years). 9 patients were eliminated from analysis because of a diagnosis of inhibitors (n=1), factor activity >1% (n=4), inadequate sample collection (n=2), patient on episodic factor replacement (n=1), and inaccurate TGA time point (n=1). The mean Factor level prior to factor administration, after elimination of the subjects (n=18) was 0.4%. As expected, our results indicate a rise in ETP and Factor activity following factor replacement, peaking at 15 minutes post infusion. The mean normalized factor clearance rate was 3.3 ± 1.2ml/h/kg. The Vdss was 2.3 ± 1 L and Factor VIII t½ was 11.5 ± 3 hours. There were strong correlations between ETP and FVIII:C (R2=0.65; p<0.0001), Peak and FVIII:C (R2=0.6; p<0.0001), R Time and Factor VIII:C (R2=0.71; p<0.0001), Peak and R Time (R2=0.59; p<0.0001), ETP and R Time (R2=0.51; p<0.0001) as shown in table 1. Table 1. Correlation data on Factor VIII:C with TGA & TEG Parameters; and TGA parameters with TEG R time R2 P-value TGA Parameters (Peak & ETP) ETP and Factor VIII:C 0.65 p<0.0001 Peak and Factor VIII:C 0.60 p<0.0001 TEG Parameter (R Time) R Time and Factor VIII:C 0.71 p<0.0001 TEG and TGA Parameters Peak and R Time 0.59 p<0.0001 ETP and R Time 0.51 p<0.0001 Conclusions: Global hemostatic assays are less expensive than traditional PK testing and are available at the time of care decisions. Results of global coagulation assays (TEG and TGA) correlated closely with FVIII activities. Global assays may predict breakthrough bleeding independent of factor levels, representing an improvement in monitoring over traditional PK. With the emergence of the bioengineered extended half-life factor products, there is a renewed interest in pharmacokinetic analysis and individualization of therapy. Assays like TEG provide the opportunity to receive feed back in real time that corresponds to FVIII activity, and enable us to make treatment decisions rapidly for each individual patient. Since these assays measure more than just the factor activity, the parameters such as ETP on TGA may be more prognostic of bleeding tendency, as has been shown previously. Pharmacokinetic and pharmacodynamics analysis of this data is ongoing. Our small sample size precludes us from making global predictions. Larger multi center trials would assist in confirming these findings. Disclosures No relevant conflicts of interest to declare.

Thrombin Generation Assay: Use in Monitoring Treatment of Patients with Coagulation Factor Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3087-3087
Author(s):  
Aine N. McCormick ◽  
Peter L. Turecek ◽  
Katalin Varadi ◽  
Geoffrey F. Savidge
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Coagulation Factor ◽  
Rational Approach ◽  
Factor Viii Inhibitor ◽  
Specific Time ◽  
Time Intervals ◽  
Monitoring Method ◽  
Standard Curve ◽  
Thrombin Generation Assay

Abstract Treatment of severe Haemophilia A relies upon the infusion of plasma derived or recombinant Factor VIII concentrates. The development of antibodies to these products is approximately 25–30% in severe cases and remains a major clinical challenge to management. Treatment in inhibitor cases with high antibody levels is based on the effect of products that bypass Factor VIII in the cascade and activate the common pathway either directly or through the extrinsic pathway. These include recombinant Factor VIIa and Factor VIII inhibitor bypassing activity (FEIBA®). At present, the use and dosage of bypassing products is severely constrained by the current inability to measure directly their beneficial effect using a standardised assay, and clinical management rests exclusively upon clinical assessment. The thrombin generation assay (TGA) provides a convenient and reproducible method for quantifying thrombin produced following activation of the cascade, and is measured by means of monitoring a fluorescent residue that arises following cleavage of an artificial thrombin substrate. Through sequential measurements quantifiable kinetic data may be collated and assessed. We have used the TGA to assess thrombin generation in inhibitor patients using samples taken at specific time intervals from patients after administration of FEIBA (n=4) or rFVIIa (n=3) following informed consent. In a number of experiments, platelets from the patient’s pre-infusion platelet rich plasma (PRP) were added. Peakthrombin (nM), peaktime (min) and maximum initial rates of thrombin generation (nM/min) from each sample were quantified, and the maximum initial rates of thrombin generation were further processed using PKAnalyst® Pharamacokinetic Data Analysis software program to define one and two PK compartment elimination models following bolus injection. Thrombin generation parameters were found to persist at levels greater than those measured for the pre infusion samples for periods beyond 6 hours post FEIBA and for 2 hours post rFVIIa, prior to reinfusion of rFVIIa. In PK evaluation studies FEIBA was found to have a relative thrombin generation T1/2 of 1–3.7 hr using the one compartment model, while rFVIIa was found to have a relative thrombin generation T1/2 of 0–2.8 hr following a single infusion. A second infusion of rFVIIa gave a relative thrombin generation T1/2 of 1.2–1.8 hr suggesting a cumulative effect. The addition of platelets from pre-infusion PRP to the assay enhanced thrombin generation by approximately 30% in all samples studied. The TGA was also used to quantify the thrombin generation produced by FEIBA and rFVIIa in samples obtained at specific time intervals post treatment, using a pre infusion sample spiked with a range of FEIBA/rFVIIa concentrations to generate a standard curve. This standard curve was then used to estimate the relative FEIBA/rFVIIa concentrations in the post infusion samples with and without the addition of platelets. We consider that the use of the TGA in this clinical setting demonstrates its value as a monitoring method highly effective for providing a rational approach to dosage adjustments of FEIBA/rFVIIa in the treatment of patients with FVIII inhibitors. This valuable analytical means for monitoring FVIII bypassing treatment is of considerable relevance in relation to surgery and the management of acute bleeds in this patient category.

scholarly journals Decreased Factor V Ameliorates Bleeding Diathesis in Patients with Combined Deficiency of Factor V and Factor VIII

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2503-2503
Author(s):  
Yanyan Shao ◽  
Wenman WU ◽  
Guanqun Xu ◽  
Wang Xuefeng ◽  
Ding Qiulan
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Similar Degree ◽  
Severe Bleeding ◽  
Factor V ◽  
Bleeding Management ◽  
Thrombin Generation Assay ◽  
The Impact

Abstract Combined factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) is a rare autosomal recessive bleeding disorder caused by mutations in LMAN1 and MCFD2 gene. Though characterized with deficiencies of both FV and FVIII, it failed to lead to more severe bleeding than single defect of FVIII in similar degree. Our study on F5F8D patients revealed that mild decrease of FV and FVIII had counteractive effect on blood coagulation. Out of 5 subjects investigated, three showed potent thrombin generation as observed in thrombin generation assay (TGA), with peak heights ranging from 138 nM to 166 nM, compared with 81 nM in normal plasma when coagulation was initiated with 1pM TF. The same trend was seen when the TF concentration was increased to 5 pM. Supplement of FV and FVIII, however, demonstrated distinct impact in TGA, with FVIII addition inducing more thrombin generation whereas higher FV level failed to further boost thrombin generation despite the shortened lag time. FV is a Janus-faced protein and suppresses blood coagulation by protecting TFPI from degradation.Similar to patients with severe FV deficiency, both total and free TFPI levels also decreased slightly in F5F8D patients. Total TFPI levels decreased from healthy donors (65.95±8.35 ng/mL) to F5F8D patients (65.79±12.99 ng/mL), to severe FV-deficient patients (51.30±11.15 ng/mL, P=0.037). A more pronounced trend was observed for free TFPI levels, as free TFPI in healthy controls were 12.68±2.91 ng/mL, higher than those in F5F8D (4.73±1.31 ng/mL, P=0.004), and in severe FV-deficient patients (3.81±1.03 ng/mL, P=0.004).The lowered TFPI in patients with F5F8D may neutralize the impact of FV and FVIII deficiency on blood coagulation. Desmopressin (DDAVP) increases endogenous FVIII and is used in the management of mild hemophilia A patients. In current study, 4 patients with F5F8D were treated with DDAVP. The administration of DDAVP saw no elevation of FV yet the increase of FVIII. The mean basal FV and FVIII levels were 11.5±3.4% and 15.1±4.8% respectively. The peak value of FVIII levels rose to 66.1±20.5% 30 minutes after the administration, and was sustained at two-folds of the baseline four hours post administration. The distinctive changes brought by combined FV and FVIII deficiencies on blood coagulation functions indicates the lone FVIII replacement without FV supplement might be sufficient in bleeding management of patients with F5F8D. DDAVP can be considered as a potential substitute for FVIII concentrate in the treatment of F5F8D patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

Blood ◽  
2011 ◽  
Vol 118 (14) ◽  
pp. 3960-3968 ◽  
Author(s):  
Kellie R. Machlus ◽  
Feng-Chang Lin ◽  
Alisa S. Wolberg
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Tissue Factor ◽  
Vascular Injury ◽  
Thrombin Generation ◽  
Elevated Plasma ◽  
Vessel Occlusion ◽  
Short Treatment ◽  
Plasma Factor

Abstract Studies have correlated elevated plasma factor VIII (FVIII) with thrombosis; however, it is unclear whether elevated FVIII is a proinflammatory biomarker, causative agent, or both. We raised FVIII levels in mice and measured the time to vessel occlusion (TTO) after ferric chloride–induced injury. Compared with control (saline-infused) mice, elevated FVIII had no effect after longer (3-minute) carotid artery injury, but it shortened the TTO after shorter (2-minute) injury (P < .008). After injury, circulating thrombin-antithrombin (TAT) complexes were lower after short versus long injury (P < .04), suggesting short treatment produced less coagulation activation. TAT levels in FVIII-infused mice were higher than in controls after short, but not longer, injury. Accordingly, elevated FVIII had no effect on in vitro thrombin generation or platelet aggregation triggered by high tissue factor, but it increased thrombin generation rate and peak (2.4- and 1.5-fold, respectively), and it accelerated platelet aggregation (up to 1.6-fold) when initiated by low tissue factor. Compared with control mice, elevated FVIII stabilized thrombi (fewer emboli) after short injury, but it had no effect after longer injury. TTO and emboli correlated with TATs. These results demonstrate dependence of FVIII activity on extent of vascular injury. We propose elevated plasma FVIII is an etiologic, prothrombotic agent after moderate but not extensive vascular damage.

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