Identification of Coagulation Factor VIII A2 Domain Residues Forming the Binding Epitope for Low-Density Lipoprotein Receptor-Related Protein<sup>,</sup>

Sarafanov, Andrey G.; Makogonenko, Yevgen; Pechik, Igor; Radtke, Klaus-Peter; Khrenov, A. V.; Ananyeva, Natalya M.; Strickland, Dudley K.; Saenko, Evgueni L.

doi:10.1021/bi0520380

Cited by 35 publications

(84 citation statements)

References 68 publications

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“…Baculovirus expressed A2 (bA2) was prepared from Sf9 cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Optimal transduction conditions were determined as described (21). The expression of bA2 was confirmed by Western blot analysis using the R8B12 antibody (25).…”

Section: Methodsmentioning

confidence: 99%

“…The second modification involved inserting the authentic residue (Gly at 374 to Val) from the A2 sequence near the N terminus of the A2. This was performed based on observations that the A2 subunit derived from the original construct showed ϳ10% of the activity as WT A2 following reconstitution analyses (21). WT and variant A2 subunits were expressed and purified using a baculovirus-directed construct from Sf9 cells as described under "Materials and Methods."…”

Section: Construction Purification and Characterization Of The Ba2mentioning

confidence: 99%

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Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

Journal of Biological Chemistry

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Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cellexpressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K d for A2. The second assay determined the K d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K d and k cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.Factor VIIIa is an essential blood coagulation protein that acts as a cofactor for the serine protease factor IXa in the conversion of factor X to Xa during the propagation phase of coagulation. Defects or deficiencies in factors VIII and IX result in hemophilia A and B, respectively. Factor VIII is synthesized as a multidomain, single chain precursor with a molecular mass of ϳ300 kDa and domain structure A1-A2-B-A3-C1-C2. It circulates primarily as a heterodimer resulting from proteolysis at the B-A3 junction wherein the heavy chain (A1A2B) and light chain (A3C1C2) are associated by metal ion-dependent and independent linkages (see Ref. 1 for review). Factor VIII is activated by limited proteolysis catalyzed by thrombin or factor Xa to the active cofactor, factor VIIIa. These cleavages convert the heterodimer or single chain precursor to the heterotrimer (2, 3) with the heavy chain-derived A1 subunit and light chain-derived A3C1C2 subunit maintaining stable association, whereas the A2 subunit is weakly associated with the A1/A3C1C2 dimer through electrostatic interactions (4, 5). Factor VIIIa can be reconstituted from the isolated A2 subunit and A1/A3C1C2 dimer to regenerate high levels of cofactor activity (3, 5).Association of factors VIIIa and IXa on a phospholipid surface forms the intrinsic factor Xase complex, which increases the cataly...

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Section: Methodsmentioning

confidence: 99%

Section: Construction Purification and Characterization Of The Ba2mentioning

confidence: 99%

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

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“…On the other hand, convincing proofs have been recently provided about interaction of FVIII with receptors of LDLR family, [44][45][46] suggesting that a prolongation of FVIII lifetime may indeed result from mutations of residues critical for the interaction of FVIII with both LRP and LDLR. 44 In conclusion, in this study 3 SNPs at the 19p3 locus (SMARCA4-LDLR) were associated with both plasma lipid concentrations and FVIII:c levels and, consistent with these intermediate phenotypes, with CAD risk. In particular, the results on rs688 suggest that an LDLR polymorphism may be associated with cardiovascular disease beyond the lipoprotein metabolism pathway, probably through a FVIII-related mechanism.…”

Section: Study Limitations and Strengthsmentioning

confidence: 99%

Polymorphisms at LDLR locus may be associated with coronary artery disease through modulation of coagulation factor VIII activity and independently from lipid profile

Martinelli

Girelli

Lunghi

et al. 2010

Blood

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High levels of coagulation factor VIII (FVIII) have been associated with cardiovascular disease. Low-density lipoprotein receptor (LDLR) has been recently demonstrated to contribute to FVIII clearance from plasma. The aim of this study was to evaluate 3 single nucleotide polymorphisms in SMARCA4-LDLR gene locus (rs1122608, rs2228671, and rs688) and FVIII coagulant activity (FVIII:c) in subjects with (n ‫؍‬ 692) or without (n ‫؍‬ 291) angiographically confirmed coronary artery disease (CAD). High FVIII:c levels were an independent risk factor for CAD. The rs688 and rs2228671 genotypes were predictors of FVIII:c with T alleles associated with higher FVIII:c levels. The rs2228671T allele was associated also with reduced total and LDL-cholesterol levels. With respect to the risk of CAD, no association was found for rs2228671. Consistently with higher FVIII:c levels, the rs688T allele was associated with CAD, whereas, consistently with a favorable lipid profile, the rs1122608T allele was associated with a decreased CAD prevalence.

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“…Interaction of FVIII with LRP involves multiple binding sites located within the A2 region 484-509 of HCh [10,32,33], the A3 region 1811-1818 of LCh, and the N-terminal portion of the C2 domain [34,35]. We have recently demonstrated that the binding sites within FVIII molecule are shared by LRP and VLDLR [14].…”

Section: Introductionmentioning

confidence: 97%

Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484–509

Ananyeva

Makogonenko

Sarafanov

et al. 2008

Blood Coagulation & Fibrinolysis

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Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays. Full-length factor VIII and its light chain bound to the receptors with similar affinities (KD = 260 +/- 9 and 156 +/- 4 nmol/l, respectively, for megalin and KD = 210 +/- 3 and 174 +/- 13 nmol/l, respectively, for sLDLR1-7). Von Willebrand factor inhibited factor VIII binding to both receptors. In contrast to the light chain, exposure of the high-affinity receptor-binding site within the heavy chain (KD = 22 +/- 4 nmol/l for megalin and 17 +/- 3 nmol/l for sLDLR1-7) required proteolytic cleavage by thrombin. This site was mapped to the A2 domain residues 484-509, based on the inhibitory effects of anti-A2 monoclonal antibody 413, and is shared by all four receptors. Using a panel of A2 mutants, we identified key amino acid residues- positively charged K466, R471, R489 and R490, and hydrophilic residues Y487 and S488- which form the frame of this 'consensus' binding site. We conclude that interaction of factor VIII with the members of the low-density lipoprotein receptor family follows the general mode, requires dissociation of factor VIII from von Willebrand factor, and is activation sensitive.

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Identification of Coagulation Factor VIII A2 Domain Residues Forming the Binding Epitope for Low-Density Lipoprotein Receptor-Related Protein^,

Cited by 35 publications

References 68 publications

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Polymorphisms at LDLR locus may be associated with coronary artery disease through modulation of coagulation factor VIII activity and independently from lipid profile

Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484–509

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Identification of Coagulation Factor VIII A2 Domain Residues Forming the Binding Epitope for Low-Density Lipoprotein Receptor-Related Protein,

Cited by 35 publications

References 68 publications

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Polymorphisms at LDLR locus may be associated with coronary artery disease through modulation of coagulation factor VIII activity and independently from lipid profile

Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484–509

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Identification of Coagulation Factor VIII A2 Domain Residues Forming the Binding Epitope for Low-Density Lipoprotein Receptor-Related Protein^,