Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Steven W. Pipe ◽  
Evgueni L. Saenko ◽  
Angela N. Eickhorst ◽  
Geoffrey Kemball-Cook ◽  
Randal J. Kaufman
Keyword(s):  
Protein Interactions ◽  
Hemophilia A ◽  
Specific Activity ◽  
Homology Model ◽  
Optical Biosensor ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Activity Assay ◽  
Subunit Dissociation ◽  
A Domains

Abstract Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, ARG531HIS, is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted ARG531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting ARG531 (ALA284GLU, ALA284PRO) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core (SER289LEU) have the same phenotype of 1-stage/2-stage discrepancy. TheALA284GLU andSER289LEU mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified ALA284GLU andSER289LEU proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the ARG531HISmutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa.

Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/ Two-stage Activity Discrepancy

2002 ◽  
Vol 88 (11) ◽  
pp. 781-787 ◽  
Author(s):  
William Hakeos ◽  
Hongzhi Miao ◽  
Nongnuch Sirachainan ◽  
Geoffrey Kemball-Cook ◽  
Evgueni Saenko ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Homology Model ◽  
Hydrophobic Core ◽  
Two Stage ◽  
Threefold Axis ◽  
Subunit Dissociation ◽  
One Stage ◽  
Fviii Activity ◽  
A Domains

SummaryThrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.

scholarly journals A topologically conserved aliphatic residue in α-helix 6 stabilizes the hydrophobic core in domain II of glutathione transferases and is a structural determinant for the unfolding pathway

1998 ◽  
Vol 336 (2) ◽  
pp. 413-418 ◽  
Author(s):  
Louise A. WALLACE ◽  
Gregory L. BLATCH ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Fluorescence Studies ◽  
Subunit Dissociation ◽  
Equilibrium Unfolding ◽  
Α Helix ◽  
Dimeric Intermediate ◽  
Conserved Residue ◽  
Structural Determinant

A topologically conserved residue in α-helix 6 of domain II of human glutathione transferase (hGST) A1-1 was mutated to investigate its contribution to protein stability and the unfolding pathway. The replacement of Leu-164 with alanine (L164A) did not impact on the functional and gross structural properties of native hGST A1-1. The wild-type protein unfolds via a three-state pathway in which only folded dimer and unfolded monomer were highly populated at equilibrium; a native-like dimeric intermediate with partially dissociated domains I and II was detected using stopped-flow fluorescence studies [Wallace, Sluis-Cremer and Dirr (1998) Biochemistry 37, 5320–5328]. In the present study, urea-induced equilibrium unfolding of L164A hGST A1-1 indicated a destabilization of the native state and suggested the presence of a stable dimeric intermediate. The unfolding kinetic pathway for L164A hGST A1-1, like that for the wild type, is biphasic, with a fast and a slow unfolding event; the cavity-forming mutation has a substantially greater effect on the rate of unfolding of the fast event. The equilibrium and kinetic unfolding data for L164A hGST A1-1 suggest that a rapid pre-equilibrium is established between the native dimer and a dimeric intermediate before complete domain and subunit dissociation and unfolding. It is proposed that the topologically conserved bulky residue in α-helix 6 plays a role in specifying and stabilizing the core of domain II and the interface of domains I and II.

scholarly journals The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 538-548 ◽  
Author(s):  
Kagehiro Amano ◽  
Rita Sarkar ◽  
Susan Pemberton ◽  
Geoffrey Kemball-Cook ◽  
Haig H. Kazazian ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Genetic Alterations ◽  
Published Data ◽  
Missense Mutations ◽  
Wild Type ◽  
Factor Ixa ◽  
Fviii Activity ◽  
A2 Domain

Abstract Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

scholarly journals Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4362-4368 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Factor Xa ◽  
Maximum Velocity ◽  
Activity Levels ◽  
Wild Type ◽  
Cleavage Rate ◽  
Type Factor ◽  
Reduced Rate

Abstract Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Michaelis-Menten constant (Km, apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (Vmax) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P1Arg, and this property likely reflects the severity of the hemophilia A phenotype.

scholarly journals The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 538-548 ◽  
Author(s):  
Kagehiro Amano ◽  
Rita Sarkar ◽  
Susan Pemberton ◽  
Geoffrey Kemball-Cook ◽  
Haig H. Kazazian ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Genetic Alterations ◽  
Published Data ◽  
Missense Mutations ◽  
Wild Type ◽  
Factor Ixa ◽  
Fviii Activity ◽  
A2 Domain

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

Whole Blood Thromboelastogram Assays Demonstrate Prolonged Factor VIIIa Potency for Recombinant Disulfide Bond-Stabilized Factor VIII Variants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2976-2976
Author(s):  
Klaus-Peter Radtke ◽  
Dean Chamberlain ◽  
John H. Griffin ◽  
Andrew J. Gale
Keyword(s):  
Disulfide Bond ◽  
Light Chain ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Disulfide Bridge ◽  
Clot Formation ◽  
Wild Type ◽  
Subunit Dissociation ◽  
A2 Domain

Abstract Following proteolytic activation of factor VIII (FVIII) by thrombin, the FVIIIa A2 domain, A3 domain and light chain (A3-C1-C2 domains) form a non-covalent hetero-trimer. Because spontaneous A2 subunit dissociation causes loss of FVIIIa activity, we previously made two mutants each with two new Cys to form a disulfide bond linking residues 662 (A2) and 1828 (A3) (FVIIIC662-C1828) or residues 664 (A2) and 1826 (A3) (FVIIIC664-C1826). Following thrombin activation, each FVIIIa mutant was stabile compared to wild type (wt) B-domain-deleted (BDD) FVIII. Previous SDS-PAGE data showed that the A2 domain was disulfide linked to the light chain. To show that this is true for undenatured FVIIIa, here we used surface plasmon resonance (SPR) to monitor A2 dissociation from thrombin-activated wild type and variant FVIII species that were bound to the sensor surface via a monoclonal antibody. Following passage of thrombin over sensor-bound FVIII, only wt FVIII showed a characteristic decrease of SPR reflecting A2 subunit dissociation and thrombin-treated FVIIIC662-C1828 and FVIIIC664-C1826 showed only minor decreases in SPR. Thus, SPR data directly demonstrate that engineered inter-domain disulfide bridges between the A2 and A3 domains prevent A2 domain dissociation from FVIIIa. In contrast to simple plasma coagulation assays of FVIIIa, rotational thromboelastogram (RoTEG) assays of whole blood provide multiple parameters reflecting clot formation, clot quality, and clot dissolution. RoTEG assays using fresh severe hemophilia A whole blood that was reconstituted with either wt FVIII, or FVIIIC662-C1828 or FVIIIC664-C1826 were performed to test the hypothesis that the disulfide-stabilized FVIIIa mutants would show improved potency for thrombin generation. After recalcification of hemophilia A blood with added FVIII, we measured the clotting time (CT), the rate of clot-formation, the clot-firmness time (CFT), defined as the time required to reach a specified clot firmness, and the clot firmness at 5 min (CF-A5), defined as the clot firmness at 5 min after the observed CT. Samples reconstituted with disulfide-bridge-stabilized FVIII mutants or wt-FVIII had comparable CTs at similar concentrations. However, in comparison to wild type BDD-FVIII, comparable rates of clot-formation, CFTs and CF-A5 were observed for up to 10-fold lower concentrations of each disulfide-bridge-stabilized FVIII mutant. The differences between wt and FVIII mutants were especially pronounced at very low FVIII concentrations whereas at FVIII concentrations >0.01 U/mL the differences were less apparent. Because clot formation occurs early relative to overall thrombin generation which is better reflected by CFT and CF-A5 values, we interpret these data to indicate that the disulfide-stabilized FVIIIa variants provide sustained thrombin generation in whole blood compared to wt FVIII and speculate that these FVIII variants may prove superior to wt FVIII for stabilizing a hemostatic plug by providing sustained thrombin generation capacity.

Inactivation-Resistant Recombinant Factor VIII Exhibits Superior Thrombin Generation Capacity in Comparison to Wild-Type and B Domain-Deleted Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1604-1604
Author(s):  
Joshua Russell ◽  
Yesim Dargaud ◽  
Randal J. Kaufman ◽  
Claude Negrier ◽  
Steven W. Pipe
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Specific Activity ◽  
Wild Type ◽  
Whitney Test ◽  
Mann Whitney Test ◽  
Transfer Strategies ◽  
The Mean

Abstract Activated factor VIII (FVIIIa) functions as a cofactor in the intrinsic hemostatic pathway leading to thrombin generation. Recombinant FVIII (rFVIII) has proven effective in replacement therapy for patients with hemophilia A (FVIII deficiency). The activity of FVIIIa in plasma is limited by both spontaneous dissociation of the A2 subunit and by cleavage by activated protein C (APC). Inactivation resistant FVIII (IR8) has been bioengineered to be resistant to both mechanisms of inactivation. The specific activity of purified IR8, as determined by one-stage clotting (aPTT) and two-stage chromogenic assays, was significantly higher (~7 to 20-fold) than that of wild-type (WT)-FVIII and B domain deleted (BDD)-FVIII. The specific activity was calculated based on ELISA antigen results and complemented by Western blots using commercial anti-FVIII antibodies. Since bioengineered IR8 may have altered immunoreactivity with anti-FVIII antibodies, an alternative functional assay was investigated to better characterize its potency. We evaluated WT-FVIII, BDD-FVIII and IR8 via the Calibrated Automated Thrombogram (CAT), a global assay of hemostasis, in platelet-free plasma (PPP) from 6 severe hemophilia A patients (<1 IU/dl FVIII) without inhibitors. The CAT test was chosen because of its ability to offer more valuable insight into the potential clinical value of IR8 than traditional clotting and chromogenic assays. Blood samples were taken into Corn trypsin inhibitor (CTI) to block contact activation and ensure that thrombin generation was triggered exclusively by tissue factor (TF) via the extrinsic hemostatic pathway. In an effort to demonstrate the dose dependency of each concentrate on its thrombin generating capacity, all 3 proteins were added to PPP along with a low TF concentration (1 pM) at varying FVIII activities (0, 25, 50 & 100 IU/dl). At each protein concentration, IR8 showed a significantly higher endogenous thrombin potential (ETP, the area under the thrombin generation curve) and peak height of the thrombin burst compared to either WT-FVIII or BDD-FVIII. The mean ETP values (nM*min) at 100 IU/dl, were WT-FVIII 650 and BDD-FVIII 725 (Mann Whitney test, p=0.69) and IR8 1107 (Mann Whitney test, p=0.04) with a mean ETP for FVIII <1% of 315 used as a control. Similar results were obtained in the presence of 1 nM thrombomodulin, which was added to sensitize the system to the action of APC. Consistent with the increased specific activity of IR8, the mean ETP of IR8 at 25 IU/dl was comparable to that of WT-FVIII at a concentration 4 times greater (100 IU/dl). Furthermore, no significant difference was found between the lag times of IR8 versus WT-FVIII and BDD-FVIII indicating that the advantage of IR8 does not lie in its ability to activate the initiation phase of thrombin generation, but rather in its persistent cofactor activity during the propagation phase of coagulation. These results are encouraging because the development of a rFVIII with markedly increased potency would potentially allow for reduced protein requirements in replacement therapy, thereby reducing costs and possibly decreasing inhibitor antibody development and would improve the efficacy of hemophilia A gene therapy without necessitating large improvements in genetic transfer strategies.

scholarly journals Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deficient Mutant Acclimated to Low and High Temperatures

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Iwona Sadura ◽  
Dariusz Latowski ◽  
Jana Oklestkova ◽  
Damian Gruszka ◽  
Marek Chyc ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Temperature Stress ◽  
High Temperatures ◽  
Biological Membrane ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Chloroplast Membranes ◽  
Paramagnetic Resonance ◽  
Photosynthetic Membranes

Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deficient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidification than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 °C, whereas in the hydrophobic core, they were visible at both 20 and 5 °C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 °C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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