A1-A2 Domain Research Articles

The impact of von Willebrand factor on fibrin formation and structure unveiled with type 3 von Willebrand disease plasma.

Normally, von Willebrand factor (VWF) remains inactive unless its A1A2 domains undergo a shear stress-triggered conformational change. We demonstrated the capacity of a recombinant A2 domain of VWF to bind and to affect fibrin formation, altering the fibrin clot structure. The data indicated that VWF contains an additional binding site for fibrin in the A2 domain that plays a role in the incorporation of VWF to the polymerizing fibrin. This study is to examine the hypothesis that active plasma VWF directly influence fibrin polymerization and the structure of fibrin clots. The study used healthy and type 3 von Willebrand disease (VWD) plasma, purified plasma VWF, fibrin polymerization assays, confocal microscopy and scanning electron microscopy. The exposed A2 domain in active VWF harbors additional binding sites for fibrinogen, and significantly potentiates fibrin formation (P < 0.02). Antibody against the A2 domain of VWF significantly decreased the initial rate of change of fibrin formation (P < 0.002). Clot analyses revealed a significant difference in porosity between normal and type 3 VWD plasma (P < 0.008), further supported by scanning electron microscopy, which demonstrated thicker fibrin fibers in the presence of plasma VWF (P < 0.0003). Confocal immunofluorescence microscopy showed punctate VWF staining along fibrin fibrils, providing visual evidence of the integration of plasma VWF into the fibrin matrix. The study with type 3 VWD plasma supports the hypothesis that plasma VWF directly influences fibrin polymerization and clot structure. In addition, a conformational change in the A1A2 domains exposes a hidden fibrin(ogen) binding site, indicating that plasma VWF determines the fibrin clot structure.

Type 2M/2A von Willebrand disease: a shared phenotype between type 2M and 2A

AbstractFour variants have been continuously subjected to debate and received different von Willebrand disease (VWD) classifications: p.R1315L, p.R1315C, p.R1374H, and p.R1374C. We chose to comprehensively investigate these variants with full set of VWD tests, protein-modeling predictions and applying structural biology. Patients with p.R1315L, p.R1315C, p.R1374H, and p.R1374C were included. A group with type 2A and 2M was included to better understand similarities and differences. Patients were investigated for phenotypic assays and underlying disease mechanisms. We applied deep protein modeling predictions and structural biology to elucidate the causative effects of variants. Forty-three patients with these variants and 70 with 2A (n = 35) or 2M (n = 35) were studied. Patients with p.R1315L, p.R1374H, or p.R1374C showed a common phenotype between 2M and 2A using von Willebrand factor (VWF):GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios and VWF multimeric profile, whereas p.R1315C represented a type 2M phenotype. There was an overall reduced VWF synthesis or secretion in 2M and cases with p.R1315L, p.R1374H, and p.R1374C, but not in 2A. Reduced VWF survival was observed in most 2A (77%), 2M (80%), and all 40 cases with p.R1315L, p.R1374H, and p.R1374C. These were the only variants that fall at the interface between the A1-A2 domains. p.R1315L/C mutants induce more compactness and internal mobility, whereas p.R1374H/C display a more extended overall geometry. We propose a new classification of type 2M/2A for p.R1315L, p.R1374H, and p.R1374C because they share a common phenotype with 2M and 2A. Our structural analysis shows the unique location of these variants on the A1-A2 domains and their distinctive effect on VWF.

Prolonged α-thrombin-related activation and delayed active protein C-associated degradation confer mild phenotype in a patient with severe hemophilia A with F8 p.H118R.

In hemophilia A, bleeding mostly correlates with factor VIII activity (FVIII:C), although some patients show discrepancy in bleeding severity and FVIII:C. We report a novel procoagulant mechanism associated with F8 p.H118R (c.353A > G) in a young Japanese man with few bleeding episodes despite low levels of FVIII:C (< 1IU/dL). Plasma FVIII:C was < 1IU/dL measured by one-stage clotting assay (OSA) and chromogenic substrate assay (CSA), whereas FVIII antigen (FVIII:Ag) was 9.7%. The global coagulation assay showed higher max speed in clot waveform analysis (CWA), shorter clotting time in rotation thromboelastometry (ROTEM) (1605 vs. > 5000s), shorter lag time (4.87 vs. 12.47min) and larger ETP (207.9 vs. 53.3nM*min) in thrombin generation assay, compared with FVIII-deficient control. Expressed recombinant H118R mutant in culture media showed low FVIII:C (1-5IU/dL) by OSA, with non-hemophilia level of FVIII:Ag. Western blot analysis using recombinant H118R showed longer persistence of heavy-chain of H118R after incubation with α-thrombin, compared with wild-type. Incubation of H118R with activated protein C (APC) also showed longer persistence of A1-A2 domain. In conclusion, H118R showed prolonged activation by α-thrombin and delayed APC-related FVIII degradation. These properties may confer the procoagulant activity and few bleeding episodes despite low FVIII:C.

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and α-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of μM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand–receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

The effect of mismatch strain induced by the a1–a2 domain pattern on dislocation generation of epitaxial ferroelectric films

This study theoretically models both mismatch strain and lattice dislocation generation induced by the a1–a2 domain pattern in epitaxial ferroelectric films. For the ferroelectric film/substrate structure, the precise complex potential functions for the Somigliana screw dislocation quadrupole that represents the lattice mismatch strain are presented, the free boundary condition of the film is approximately processed by an equivalent method, and the effect of mismatch strain on lattice dislocation generation is discussed. The results show that: the higher mismatch degree and domain wall energy evidently lead to interfacial dislocation multiplication; the density of the dislocations decreases with decreasing film thickness; the generated dislocations are mainly concentrated at the sites far away from domain walls.

Hierarchy and scaling behavior of multi-rank domain patterns in ferroelectric K0.9Na0.1NbO3 strained films

The formation process of a ferroelectric multi-rank domain pattern in the thickness range of 7–52 nm is investigated for monoclinic K0.9Na0.1NbO3 strained epitaxial films on (110) NdScO3 substrates. Although the elastic strain energy density is degenerated for two pseudocubic orientations, a distinctive hierarchy of domain evolution is observed with exclusive in-plane a1a2 domains for very thin films and the retarded onset of a ferroelectric MC phase at larger film thickness. This is accompanied by a thickness dependent transformation from stripe domains to a herringbone pattern and, eventually, for the thickest film, to a checkerboard-like structure. These transformations in the domain arrangement and width are correlated to energetic aspects as depolarization field and anisotropic strain relaxation in the film. While for the MC domains plastic strain relaxation is throughout observed, the a1a2 domains show a two-step strain relaxation mechanism starting with an in-plane elastic shearing, which is followed by plastic lattice relaxation. Our results highlight a pathway for engineering and patterning of periodic ferroelectric domain structures.

Strain Engineering of Ferroelectric Domains in KxNa1−xNbO3 Epitaxial Layers

The application of lattice strain through epitaxial growth of oxide films on lattice mismatched perovskite-like substrates strongly influences the structural properties of ferroelectric domains and their corresponding piezoelectric behavior. The formation of different ferroelectric phases can be understood by a strain-phase diagram, which is calculated within the framework of the Landau-Ginzburg-Devonshire theory. In this paper, we illustrate the opportunity of ferroelectric domain engineering in the KxNa1-xNbO3 lead-free material system. In particular, the following examples are discussed in detail: (i) Different substrates (NdGaO3, SrTiO3, DyScO3, TbScO3, GdScO3) are used to systematically tune the incorporated epitaxial strain from compressive to tensile. This can be exploited to adjust the NaNbO3 thin film surface orientation, and, concomitantly, the vector of electrical polarization, which rotates from mainly vertical to exclusive in-plane orientation. (ii) In ferroelectric NaNbO3 thin films grown on rare-earth scandate substrates, highly regular stripe domain patterns are observed. By using different film thicknesses, these can be tailored with regard to domain periodicity and vertical polarization component. (iii) A featured potassium concentration of x = 0.9 of KxNa1 xNbO3 thin films grown on (110) NdScO3 substrates favors the coexistence of two equivalent, monoclinic, but differently oriented ferroelectric phases. A complicated herringbone domain pattern is experimentally observed which consists of alternating MC and a1a2 domains. The coexistence of different types of ferroelectric domains leads to polarization discontinuities at the domain walls, potentially enabling high piezoelectric responses. In each of these examples, the experimental results are in excellent agreement with predictions based on the linear elasticity theory.

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97-125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83-145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1-2 nm). However, addition of two PAM residues (Arg(126)-His(127)) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg(113), His(114), Glu(116), Arg(126), His(127)), mutation of which reduced PAM binding affinity for K2hPg by ∼ 1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.

Effects of replacement of factor VIII amino acids Asp519 and Glu665 with Val on plasma survival and efficacy in vivo.

Proteolytic cleavage of factor VIII (FVIII) to activated FVIIIa is required for participation in the coagulation cascade. The A2 domain is no longer covalently bound in the resulting activated heterotrimer and is highly unstable. Aspartic acid (D) 519 and glutamic acid (E) 665 at the A1-A2 and A2-A3 domain interfaces were identified as acidic residues in local hydrophobic pockets. Replacement with hydrophobic valine (V; D519V/E665V) improved the stability and activity of the mutant FVIII over the wild-type (WT) protein in several in vitro assays. In the current study, we examined the impact of mutations on secondary and tertiary structure as well as in vivo stability, pharmacokinetics (PK), efficacy, and immunogenicity in a murine model of Hemophilia A (HA). Biophysical characterization was performed with far-UV circular dichroism (CD) and fluorescence emission studies. PK and efficacy of FVIII was studied following i.v. bolus doses of 4, 10 and 40 IU/kg with chromogenic and tail clip assays. Immunogenicity was measured with the Bethesda assay and ELISA after a series of i.v. injections. Native secondary and tertiary structure was unaltered between variants. PK profiles were similar at higher doses, but at 4 IU/kg plasma survival of D519V/E665V was improved. Hemostasis at low concentrations was improved for the mutant. Immune response was similar between variants. Overall, these results demonstrate that stabilizing mutations in the A2 domain of FVIII can improve HA therapy in vivo.

The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.

A Novel Molecular Mechanism In The Interplay Of Platelet GPIb-VWF-Fibrin In Thrombus Formation

It is established that the interplay between von Willebrand factor (VWF) and fibrinogen is relevant for the platelet-platelet interaction in thrombus formation. Although the concentration of fibrinogen is much higher than that of VWF in blood, normally, they do not interact. In fact, VWF in plasma has a poor binding activity for fibrin(ogen)-coated surface, which exposes fibrin-specific sequences when it is immobilized. This observation suggests that 1) the site recognized by fibrin is inaccessible in plasma VWF, and 2) a conformational change in the VWF protein is a pre-requisite to bind fibrin. The latter may be supported by a previous study indicating that VWF has to bind to its platelet receptor glycoprotein (GP)Ib first in order to bind fibrin. Another line of evidence suggests that the binding of VWF to the polymerizing fibrin facilitates the interaction of platelet GPIb with the fibrin-bound VWF during the growth of the thrombus, a mechanism apparently defective in Bernard-Soulier syndrome (BSS) and von Willebrand disease (VWD). Consistent with this, fibrin monomer, like ristocetin, can induce the binding of VWF to GPIb. The C1C2 domains have been described as the fibrin-binding domain in VWF, and it is unknown whether the structures of these domains change to bind to fibrin. In contrast, conformational changes within the A1A2 domains of VWF induced by ristocetin result in the binding to GPIb and the protease ADAMTS-13. Therefore, we investigated a potential contact site for fibrin among the A1A2 domains of VWF. We characterized the interaction of each plasma (p)VWF, purified pVWF, recombinant A1A2A3 domains protein, single A1, and single A2 domains with fibrinogen, fibrin, or D-dimer. We also examined the effect of ristocetin in the VWF-fibrin binding. Both pVWF and purified pVWF bound to immobilized fibrin(ogen) in a ristocetin dependent manner. An A1A2A3 mutant expressing increased GPIb-binding activity bound to fibrin(ogen) in the absence of ristocetin. Both A1 and A2 domains, but not the A3 domain had binding activity for immobilized fibrin(ogen). Both soluble fibrin monomer and D-dimer, unlike fibrinogen in solution, had a significant binding activity for the immobilized A2 domain. Moreover, the A2 domain was capable of delaying fibrin polymerization in vitro. The use of conformation-specific monoclonal antibodies demonstrated that, like collagen, fibrin(ogen) induces a change in the structure of the A1 domain, and platelets interacted effectively with the fibrin(ogen)-bound A1 domain under high shear stress. Interestingly, in contrast to A3 or A1domain, flowing platelets did not adhere to the fibrin(ogen)-coated surface incubated with the A2 domain. In conclusion, we have demonstrated a previously unknown fibrin(ogen)-binding activity for the A1 and A2 domains in VWF. These observations reveal new mechanisms that may help to understand the pathophysiology of the bleeding in VWD, in which a number of natural occurring mutations that cause the disease are found within the A1 and A2 domains, and the poor fibrin polymerization observed in BSS. Furthermore, these outcomes may have implications for a better understanding of the high risk for thrombosis when the levels of both VWF and fibrinogen are increased. Disclosures:No relevant conflicts of interest to declare.

Modification of Interdomain Interfaces within the A3C1C2 Subunit of Factor VIII Affects Its Stability and Activity

Factor (F)VIII consists of a heavy chain [A1(a1)A2(a2)B domains] and a light chain [(a3)A3C1C2 domains]. Several reports have shown significant changes in FVIII stability and/or activity following selected mutations at the A1-A2, A1-A3, A2-A3, and A1-C2 domain interfaces. In this study, the remaining inter-FVIII subunit interfaces (A3-C1 and C1-C2) were examined for their contributions to the stability and activity of FVIII and FVIIIa. We prepared FVIII mutants with nascent disulfide bridges between A3 and C1 domains (Gly1750Cys/Arg2116Cys and Ala1866Cys/Ser2119Cys) or C1 and C2 domains (Ser2029Cys/Pro2292Cys). We also prepared mutants via replacement of Arg2116 with hydrophobic residues (Ala and Val) because this C1 domain residue appears to face a pocket of positive electrostatic potential in the A3 domain. Stability was assessed following the rates of loss of FVIII activity at 55 °C and the spontaneous loss of FVIIIa activity from A2 subunit dissociation. FVIII Gly1750Cys/Arg2116Cys showed a marked increase in thermal stability (∼3.7-fold) compared with that of wild-type (WT) FVIII, while the stability of FVIII Ala1866Cys/Ser2119Cys was reduced (∼4.7-fold). Although the Ser2029Cys/Pro2292Cys variant showed a modest loss of FVIII stability, the specific activity and thrombin generation potential of this variant were increased (up to 1.2-fold) compared with those of WT. Furthermore, this variant demonstrated an ∼2-fold reduced Km for FX. Mutation of Arg2116 to hydrophobic residues resulted in variable decreases in stability and thrombin generation parameters, suggesting a role of this Arg residue contributing to FVIII structure. Taken together, selective modification of the contiguous domain interfaces in the FVIII light chain may improve FVIII stability and/or cofactor function.

The diversity of the immune response to the A2 domain of human factor VIII

AbstractApproximately 30% of patients with severe hemophilia A develop inhibitory anti–factor VIII (fVIII) antibodies (Abs). We characterized 29 anti-human A2 monoclonal Abs (mAbs) produced in a murine hemophilia A model. A basis set of nonoverlapping mAbs was defined by competition enzyme-linked immunosorbent assay, producing 5 major groups. The overlapping epitopes covered nearly the entire A2 surface when mapped by homolog-scanning mutagenesis. Most group A mAbs recognized a previously described epitope bounded by Arg484-Ile508 in the N-terminal A2 subdomain, resulting in binding to activated fVIII and noncompetitive inhibition of the intrinsic fXase complex. Group B and C mAbs displayed little or no inhibitory activity. Group D and E mAbs recognized epitopes in the C-terminal A2 subdomain. A subset of group D mAbs inhibited the activation of fVIII by interfering with thrombin-catalyzed cleavage at Arg372 at the A1-A2 domain junction. Other group D mAbs displayed indeterminate or no inhibitory activity despite inhibiting cleavage at Arg740 at the A2-B domain junction. Group E mAbs inhibited fVIII light-chain cleavage at Arg1689. Inhibition of cleavages at Arg372 and Arg1689 represent novel mechanisms of inhibitor function and, along with the extensive epitope spectrum identified in this study, reveal hitherto unrecognized complexity in the immune response to fVIII.

Studies on the Essential Intramolecular Interaction Between the A1 and A2 Domains of von Willebrand Factor

Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region. Keywords: von Willebrand factor, A1-A2 domain interaction, Circular Dichroism spectroscopy, Molecular Dynamics simulations

Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ∼3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ∼4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ∼4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.

Large electric field induced strains in ferroelectric islands

An effective mechanism that generates large recoverable electric field induced strains in ferroelectric islands is studied by phase-field modeling. The large strains originate from the reversible 90° domain switching between a1 (a2) domains and c domains, driven by an applied electric field and an internal stress field. The electric field induced strains could be effectively controlled by the magnitude of island-substrate misfit and the aspect ratio of islands.

Mechanisms of Human Neutrophil Elastase-Catalyzed Inactivation of the Factor VIII(a).

Abstract 2125Poster Board II-103Inflammation and coagulation are linked in a variety ways. Since both neutrophil elastase and cathepsin G bind to activated platelets, they can be localized on the platelet membrane providing negatively-charged phospholipid, that is essential for assembly of tenase complex. Although it has been reported that cathepsin G provides some procoagulant effect by activating factor (F)VIII, effect of elastase on FVIII is poorly understood. We now examine the effect of human neutrophil elastase (HNE) on FVIII(a). FVIII activity (used 100 nM) was rapidly decreased and was undetectable within 10 min after the addition of HNE (0.5 nM) in a one-stage clotting assay. This time-dependent inactivation was little or mildly affected (∼35% inhibition at 10 μg/ml) in the presence of phospholipid or von Willebrand factor, respectively. Rate constant of FVIII inactivation by HNE possessed ∼15-fold greater than that by APC/protein S (PS). SDS-PAGE analysis revealed that HNE proteolyzed the 90∼210-kDa heavy chain of FVIII to 47- and 37-kDa terminal products, via some proteolyses within the B domain. Western blot and N-terminal sequence analyses showed that these fragments derived from the heavy chain were identified as the A11-357 and A2375-708, respectively, by cleavages at Val357 and Val374 in A1 and Val708 in A2. The cleavages at Val374 and Val708 by HNE were faster than that at Val357. The 80-kDa light chain was proteolyzed to 75-kDa fragment by cleavage at Val1670, quite different with the pattern of FVIII cleavage by known serine proteases. Of note, HNE-catalyzed inactivation of FVIIIa was much slower (by ∼25-fold) than that of FVIII, supporting that HNE preferably attacked to FVIII. This discrepancy was attributed to much slower cleavage at Val708 in FVIIIa. Rate of inactivation of FVIIIa by HNE was ∼30-, ∼8-, and ∼3-fold lower than those by plasmin, APC/PS, and FXa, respectively. SDS-PAGE using FVIII(a) fragments revealed that each cleavage at Val357 and Val708 was regulated by the presence of the light chain and heavy chain (intact A1-A2), respectively. These results demonstrate that the importance of cleavage at Val708, constitutes a FIXa-interactive site in tenase complex, for the mechanism of HNE-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A1-A2 domain that might interact with HNE. Disclosures:No relevant conflicts of interest to declare.

Increasing Hydrophobicity and Disulfide Bridging Between A1 and C2 Subunits Improves Factor VIII Stability.

Abstract 3176Poster Board III-100Factor VIII (FVIII) consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Previously we have generated FVIII mutants with enhanced stability by mutating residues located at A1-A2 or A2-A3 interfaces (Wakabayashi et al, Blood, 112, 2761-9, 2008, Wakabayashi et al, J. Thromb. Haemost. 7, 438-44, 2009). FVIII X-ray structures show close contacts between the Ca2+ binding site contained within the A1 domain and the C2 domain of LC. In this study we mutated residues located at this interface to examine the effects on FVIII(a) stability. Studies assessing FVIII thermal and chemical stability involved monitoring the rates of loss of FVIII activity by FXa generation assay following incubation of FVIII (4 nM) at 57°C or in various concentrations of guanidinium (0-1.2 M). The rate of decay of FVIIIa was monitored over time at 23°C using FXa generation assays following activation of FVIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. In one variant, a disulfide bond was introduced between the two domains by a double mutation at Arg121 in A1 and Leu2302 in the C2 domain to Cys (R121C/L2302C). In addition, based on the finding that there is a gap between the methyl groups of Ala108 (A1 domain) and Ala2328 (C2 domain) we mutated Ala108 to Val, Ile, or Leu to examine whether these mutants increase the stability of FVIII by an improved hydrophobic interaction at this site. Significant increases in FVIII thermal stability, up to 4-fold compared with WT, were observed in R121C/L2302C, Ala108Ile, and Ala108Leu. R121C/L2302C and Ala108Ile retained ∼80% FVIII activity as measured by FXa generation assay compared to WT value, however, that of Ala108Leu was ∼25% the WT value. Only Ala108Ile showed an improvement in chemical stability (10% increase in IC50 value as compared with WT FVIII) and FVIIIa decay due to A2 subunit dissociation was similar to WT FVIII (20-40% reduction in FVIIIa decay rate compared to WT). Ca2+ is necessary for FVIII function and EGTA (2 mM) reduced WT FVIII activity by ∼70%. However, EGTA-treated R121C/L2302C FVIII retained ∼100% activity, suggesting that the Ca2+ requirement for FVIII function may be substituted by covalent bonding between the Ca2+ binding region in A1 and C2 subunit. Furthermore, the Ala108Ile variant showed ∼60% activity remaining after EGTA treatment suggesting partial relief of this Ca2+ dependency for stability of the A1-C2 interaction. Next, we tested whether the mutations at the A1-C2 interface can be combined with mutations at A1-A2 or A2-A3 interfaces to generate a FVIII with further improved stability. Previously characterized FVIII variants, designated A domain mutants, showing up to 2-fold increases in thermal stability compared with WT FVIII included Asp519Ala, Asp519Val, Glu665Ala, Glu665Val, Glu1984Ala, and Glu1984Val. In combining those mutations with either R121C/L2302C or Ala108Ile, we obtained variants with >5-fold increases in thermal stability (9/12 mutants), with the Ala108Ile/Glu665Val variant showing the greatest increase (∼10-fold). Most of the mutants (9/12) showed normal FVIII activity values by FXa generation assay (>60%) and 15-30% increases in IC50 values for chemical stability as compared with WT. In addition, the high FVIIIa stability of the A domain mutants was largely preserved in the combined mutations. Collectively, these results suggest that alterations at this A1-C2 contact region by covalent modification or increasing hydrophobic interaction yields improved FVIII stability that can be combined with other high stability mutations to produce additive effects. DisclosuresNo relevant conflicts of interest to declare.

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations.

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 112, 2761, 2008). In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 degrees C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L(-1)) FVIII concentration. Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.