Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit

SummaryFactor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.

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Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

Thrombosis and Haemostasis ◽

10.1160/th13-10-0918 ◽

2014 ◽

Vol 112 (07) ◽

pp. 43-52 ◽

Cited By ~ 1

Author(s):

Hironao Wakabayashi ◽

Jennifer M. Wintermute ◽

Philip J. Fay

Keyword(s):

Thermal Stability ◽

Thrombin Generation ◽

Resistant Mutant ◽

Decay Rates ◽

Single Type ◽

Wild Type ◽

Subunit Interactions ◽

Factor Viiia ◽

The Stability ◽

A3 Subunit

SummaryFVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a) variants. Separately we showed that altering the sequence flanking the primary FXa cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3’)562) combined with mutations of Ala108Ile, Asp519Val/ Glu665Val or Ala108Ile/Asp519Val/Glu665Val and examined the effects of these combinations relative to FVIII thermal stability, rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay rates for 336(P4-P3’)562/Ala108Ile, 336(P4-P3’)562/Asp519Val/ Glu665Val, and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼2– to 5-fold as compared with wild-type (WT) primarily reflecting the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼25 fold, indicating greater stability than the control Asp519Val/Glu665Val variant (∼14-fold). Interestingly, 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/ Asp519Val/Glu665Val variants showed reduced FXa-inactivation rates compared with the 336(P4-P3’)562 control (∼4-fold), suggesting A2 subunit destabilisation is a component of proteolytic inactivation. Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val control. These results indicate that combining multiple gain-of-function FVIII mutations yields FVIII variants with increased stability relative to a single type of mutation.

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Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽

10.1182/blood.v110.11.2686.2686 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2686-2686

Author(s):

Jennifer Newell ◽

Qian Zhou ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Specific Activity ◽

Factor Xa ◽

Factor X ◽

Wild Type ◽

Factor Viiia ◽

N Terminus ◽

Acidic Residues ◽

Type Factor ◽

A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

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Partial Reconstitution of Factor VIII Activity from a Mild Crm+ Hemophilia A Patient by Replacement of the Defective A2 Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615099 ◽

1998 ◽

Vol 79 (05) ◽

pp. 943-948 ◽

Cited By ~ 1

Author(s):

W. C. Pieneman ◽

P. Fay ◽

E. Briët ◽

P. H. Reitsma ◽

R. M. Bertina

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Wild Type ◽

Coding Region ◽

Factor Viii Activity ◽

Factor Ixa ◽

Factor Viiia ◽

Factor Viii Antigen ◽

A2 Domain ◽

Reduced Activity

SummaryWe further characterised the abnormal factor VIII molecule (factor VIII Leiden) of a Crm+, mild hemophilia A patient with a factor VIII activity of 0.18 IU/ml and a factor VIII antigen of 0.95 IU/ml. Mutation analysis of the coding region, promoter and 3’ untranslated region of the factor VIII gene revealed the presence of a C to T substitution at codon 527. This nucleotide change predicts the replacement of an arginine to tryptophan in the A2 domain close to a suggested binding site for factor IXa. Since a previous study of this mutant factor VIII protein suggested that this protein had a reduced affinity for factor IXa, position 527 in the protein might be involved in the interaction with factor IXa.In this study we gathered evidence for our hypothesis that the Arg to Trp mutation at position 527 is the cause of the reduced activity of factor VIII Leiden. Replacement of the mutated A2 domain by wild type A2 domain partially corrected the defect.Factor VIII from normal and factor VIII Leiden plasma was concentrated by cryoprecipitation, activated with thrombin and incubated with excess wild type A2 domain. Competition with excess isolated human A2 domain resulted in a partial reconstitution of the factor VIIIa activity of thrombin treated factor VIII Leiden. This supports the hypothesis that the mutation in the A2 domain is the cause of the reduced factor VIII activity.

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Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Blood ◽

10.1182/blood-2008-02-142158 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2761-2769 ◽

Cited By ~ 28

Author(s):

Hironao Wakabayashi ◽

Fatbardha Varfaj ◽

Jennifer DeAngelis ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Specific Activity ◽

Fold Increase ◽

Covalent Modification ◽

Stability Factor ◽

Wild Type ◽

Factor Viiia ◽

Charged Residues ◽

A2 Domain ◽

Enhanced Stability

AbstractFactor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.

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Computational ligands to VKORC1s and CYPs. Could they predict new anticoagulant rodenticides?

10.1101/2021.01.22.426921 ◽

2021 ◽

Author(s):

A Bermejo-Nogales ◽

J.M. Navas ◽

J Coll

Keyword(s):

Experimental Validation ◽

Molecular Mechanisms ◽

Computational Studies ◽

Binding Affinities ◽

Wild Type ◽

Anticoagulant Rodenticide ◽

High Binding ◽

Related Enzyme ◽

Resistant Mutants ◽

Target Effects

AbstractAnticoagulant-resistance in rodents and anticoagulant off-target effects are some of the world-wide problems of increasing concern. To search for new anticoagulant rodenticide candidates we have computationally explored some of the rat genes previously implicated in resistance to actual anticoagulants. In particular, we searched among hundreds of anticoagulant-similar chemotypes those binding rat wild-type VKORC1 (the best-known anticoagulant target, a Vitamin K-recycling enzyme), VKORC1L1 (a VKORC1-related enzyme), Cytochrome P450 CYP enzymes (some of the most important enzymes implicated in detoxification) and anticoagulant-resistant VKORC1-mutants (to minimize propensity to resistance). Results predicted new VKORC1 leads with binding-scores in the low nM range (high binding-affinities) predicting hydroxycoumarin- and naphtoquinone-like chemotypes. We then selected top-leads with additional high binding-scores to more than three anticoagulant-related CYPs, suggesting minimal detoxification rates and therefore maximal anticoagulation expectatives. A downsized list of top top-leads maintaining VKORC1 low-binding scores to anticoagulant resistant mutants, was finally proposed for experimental validation. The combination of different rat targets for computational studies, could be used to search for unrelated chemotypes, for reduction of off-target environmental anticoagulant impacts, and/or as new tools to explore anticoagulant molecular mechanisms.

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

Blood ◽

10.1182/blood.v104.11.2976.2976 ◽

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.

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IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

Molecular Biology of the Cell ◽

10.1091/mbc.e09-04-0277 ◽

2009 ◽

Vol 20 (13) ◽

pp. 3055-3063 ◽

Cited By ~ 43

Author(s):

Raqual Bower ◽

Kristyn VanderWaal ◽

Eileen O'Toole ◽

Laura Fox ◽

Catherine Perrone ◽

...

Keyword(s):

Double Mutant ◽

Essential Role ◽

Null Allele ◽

Flagellar Motility ◽

Wild Type ◽

Gene Encoding ◽

Radial Spoke ◽

Mutant Strains ◽

Dynein Arms ◽

Image Averaging

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

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Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Eukaryotic Cell ◽

10.1128/ec.00040-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1475-1485 ◽

Cited By ~ 33

Author(s):

Thanyanuch Kriangkripipat ◽

Michelle Momany

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Elevated Temperatures ◽

Cell Wall Integrity ◽

Wild Type ◽

Spatial Cues ◽

Developmental Patterning ◽

In The Wild ◽

Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

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A Reference Scale of Cucurbit[7]uril Binding Affinities

Organic & Biomolecular Chemistry ◽

10.1039/d1ob01304a ◽

2021 ◽

Author(s):

Mohammad A. Alnajjar ◽

Werner M Nau ◽

Andreas Hennig

Keyword(s):

Accurate Determination ◽

Binding Affinities ◽

High Binding ◽

Reference Scale ◽

Direct Binding ◽

Guest Chemistry

The accurate determination of ultra-high binding affinities in supramolecular host-guest chemistry is a challenging endeavour because direct binding titrations are generally limited to affinities <106 M-1 due to sensitivity constraints...

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