Identification of a Plasmin-Interactive Site within the A2 Domain of the Factor VIII Heavy Chain.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1018-1018 ◽  
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
Katsumi Nishiya ◽  
Evgueni L. Saenko ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Rate Constant ◽  
Heavy Chain ◽  
Active Site ◽  
Inactivation Rate ◽  
Wild Type ◽  
Inactivation Rate Constant ◽  
Factor Ixa ◽  
A2 Domain

Abstract Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991, BLOOD102, 2002). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by <20%). To investigate the responsible residues in A2 domain for plasmin binding, the mutant forms of the A2 domain were expressed in baculovirus system and purified. Compared with wild type (23 nM), the affinity of R484A mutant was dramatically decreased by ~250-fold, and the affinities of K377A, K466A, R471A, and K523A mutants also were decreased by 10~40-folds, respectively. Especially, the addition of R484A mutant was reduced inactivation rate constant of plasmin-catalyzed FVIIIa by only ~40% of that of wild type. These findings demonstrate that Arg484 in the A2 domain contains plasmin-binding site responsible for plasmin-catalyzed FVIII(a) inactivation.

Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1711-1711 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Timothy Myles ◽  
Lawrence L. Leung ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Active Site ◽  
Specific Activity ◽  
Solid Phase ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Marginal Effect ◽  
Thrombin Cleavage ◽  
A2 Domain

Abstract Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

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

1998 ◽  
Vol 79 (05) ◽  
pp. 943-948 ◽  
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.

scholarly journals Cleavage of Factor VIII Heavy Chain Is Required for the Functional Interaction of A2 Subunit with Factor IXa

2001 ◽  
Vol 276 (15) ◽  
pp. 12434-12439 ◽  
Author(s):  
Philip J. Fay ◽  
Maria Mastri ◽  
Mary E. Koszelak ◽  
Hironao Wakabayashi
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Fluorescence Anisotropy ◽  
Factor Xa ◽  
Factor X ◽  
Functional Interaction ◽  
Factor Ixa ◽  
Factor Viiia ◽  
A2 Domain ◽  
Stimulation Of

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

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 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.

Substitution of Four Residues Revealed by High-Throughput Screening of Human Factor VIII A2 Domain Generated a Recombinant Molecule That Escapes to Anti-Factor VIII Antibodies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3067-3067
Author(s):  
Jean-Luc Plantier ◽  
Didier Saboulard ◽  
Marc Delcourt ◽  
Nathalie Enjolras ◽  
Claude Negrier
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
High Throughput ◽  
Solid Phase ◽  
Binding Capacity ◽  
Residual Activity ◽  
Wild Type ◽  
Human Factor Viii ◽  
Inhibitory Antibodies ◽  
A2 Domain

Abstract Using Massive Mutagenesis technique® we performed a high-throughput alanine substitution of 206 residues between aminoacids 376 to 649 from factor VIII (FVIII) A2 domain. The pattern of activity and the levels of production of FVIII mutants were assessed following transient expression in COS-1 cells. FVIII mutants that kept at least 50% of wild-type activity were then screened in an inhibitor assay against total immunoglobulin G (IgG) fractions from patients with severe hemophilia A who had developed inhibitory antibodies (n=4; range 6–15 BU/mL) or a non immune IgG as control. In this assay, the cell culture supernatants containing FVIII were incubated in a volume of FVIII-depleted plasma for 1h30 in the presence of IgG. The residual activity was then measured in a chronometric assay. No single mutations were able to significantly allow FVIII to escape inhibitors. Four mutations (S409A, L462A, E507A, L629A) having a tendency to resist to inhibitors were selected and recombined two by two leading to a significant but insufficient resistance to anti-FVIII antibodies. The effect of the mutations was additive since a molecule (FVIII-4A2) combining the 4 substitutions significantly resisted to the inhibitory antibodies. Residual activity of FVIII-4A2 ranged from 8% to up to 82% of the initial activity depending on the inhibitor plasma whereas this residual activity never exceeded 30% for control wild-type FVIII. Following production by CHO cells, purified FVIII-4A2 demonstrated a similar pattern of resistance to the four IgG fractions already assayed. FVIII-4A2 was then assayed against 11 additional unrelated inhibitors (range 3–2662 BU/mL) and displayed also a resistance against 10 out of the 11 IgG fractions. The resistance was in all case only partial in relation with the likely presence of anti-C2 and/or anti-A3-C1 inhibitors within the IgG fractions. As detected in a solid-phase assay, the decrease in inhibitory effect was for some of the IgG fractions partly related to a decrease in their binding capacity. As a control experiment, FVIII-4A2 was poorly recognized by the monoclonal antibody GMA012 directed against the A2 domain. In contrast, the binding to ESH4, an anti-C2 monoclonal antibody was not affected. Such combination of mutations opened the perspective for the generation of a recombinant FVIII molecule that can be used as an effective substitutive FVIII therapy in patients with inhibitors.

Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

2021 ◽  
Author(s):  
Yuto Nakajima ◽  
Hiroaki Minami ◽  
Keiji Nogami
Keyword(s):  
Surface Plasmon Resonance ◽  
Factor Viii ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
C2 Domain ◽  
Wild Type ◽  
Cross Link ◽  
Domain Specific ◽  
Cofactor Activity ◽  
Acidic Region

AbstractFactor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.

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

Blood ◽  
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.

scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

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