共查询到20条相似文献,搜索用时 43 毫秒
1.
Patrick H. N. Celie Gunny van Stempvoort Sylvie Jorieux Claudine Mazurier Jan A. van Mourik & Koen Mertens 《British journal of haematology》1999,106(3):792-800
The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527. 相似文献
2.
Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine 总被引:10,自引:4,他引:6
The effective activation of factor X by factor IXa requires the co-factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303-2317, 2305-2332, and 2308-2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318-2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid. 相似文献
3.
The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIII(WT) and FVIII(Arg562Ala) showed catalytic rate constant (k(cat)) values of approximately 60 minute(-1) in the presence of phospholipid, whereas the hemophilia A-associated mutants showed k(cat) values ranging from 3.3 minute(-1) to 7.5 minute(-1). In the absence of phospholipid, all k(cat) values were reduced but FVIII(WT) and FVIII(Arg562Ala) retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa. 相似文献
4.
Nogami K Shima M Giddings JC Takeyama M Tanaka I Yoshioka A 《International journal of hematology》2007,85(4):317-322
Some factor VIII (FVIII) inhibitor alloantibodies block FVIII binding to von Willebrand factor (VWF) and phospholipid (PL) and recognize a C2 domain epitope that overlaps both binding sites. We previously showed that FVIII peptide 2315-2330 neutralized FVIII inhibitors and that Cys2326 and Glu2327 contributed to the maximum neutralizing effect. In the present study, we investigated the relationship between the essential binding sites for VWF, PL, and anti-C2 inhibitors by means of competitive-inhibition assays with overlapping synthetic peptides that span the C terminus of the C2 domain (residues 2288-2332). We identified 2 peptides (residues 2303-2317 and 2315-2330) that specifically blocked FVIII binding to VWF or PL by approximately 80% (50%-inhibitory concentration [IC50], 9.0 microM) and 95% (IC50, 0.12 microM), respectively. To examine in detail the residues responsible for PL binding, we prepared mutants of peptide 2315-2330 in which we sequentially substituted each residue with Gly. Two residues, Ile2317 and Met2321, were shown to be essential for PL binding. Their substitution with Gly reduced the inhibitory effect by >90%. The data suggest that the binding sites for VWF, PL, and anti-C2 inhibitors in the C2 domain are in very close proximity but are not identical. 相似文献
5.
Keiji Nogami Midori Shima Hiroaki Nakai Ichiro Tanaka Hiroshi Suzuki Shogo Morichika Masaru Shibata Evgueni Saenko Dorothea Scandella John Giddings & Akira Yoshioka 《British journal of haematology》1999,107(1):196-203
Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3-C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy-terminal region of the C2 domain (residues 2288-2332). We found one peptide (2315-2330, L9) which neutralized the anti-FVIII activity of four out of five different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173-2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218-2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315-2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg2320 and Cys2326 or Glu2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes. 相似文献
6.
A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C-catalyzed inactivation 总被引:3,自引:0,他引:3
Nogami K Shima M Nishiya K Hosokawa K Saenko EL Sakurai Y Shibata M Suzuki H Tanaka I Yoshioka A 《Blood》2002,99(11):3993-3998
The protective effect of von Willebrand factor (VWF) toward activated protein C (APC)-catalyzed inactivation of factor VIII (FVIII) has been attributed mainly to inhibition of FVIII binding to phospholipid. In the present study, we demonstrated that VWF-mediated FVIII protection from APC also results from direct inhibition of FVIII binding to APC. Inhibition of FVIII binding to anhydro-APC by VWF would be consistent with partial or complete overlap of the FVIII binding sites for APC and VWF. We examined, therefore, the inhibitory effects of 6 synthetic peptides spanning residues 1996 to 2028 around the previously localized APC binding region (FVIII residues 2009-2018). Peptide 2009 to 2018 inhibited FVIII binding to anhydro-APC by 83% (50% inhibition, 55 microM). Similarly, peptide 2013 to 2022 inhibited FVIII binding to VWF by 84% (50% inhibition, 25 microM). It was also found that peptides 2009 to 2018 and 2013 to 2022 optimally bound to anhydro-APC and VWF, respectively. A rabbit antipeptide IgG, raised against peptide 2009 to 2022, blocked the binding of both anhydro-APC and VWF to FVIII. This immunoglobulin G inhibited proteolytic cleavage of FVIII by APC. Our results indicate that the essential regions for the binding of APC and VWF to FVIII overlap and that the protective effect of VWF on APC-catalyzed FVIII inactivation includes competitive inhibition of APC binding to FVIII by VWF. 相似文献
7.
van Den Brink EN Turenhout EA Davies J Bovenschen N Fijnvandraat K Ouwehand WH Peters M Voorberg J 《Blood》2000,95(2):558-563
A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by V(H) domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the V(H)1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study. (Blood. 2000;95:558-563) 相似文献
8.
Common inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor 总被引:4,自引:0,他引:4
Midori Shima Hiroaki Nakai Dorothea Scandella Ichiro Tanaka Yoshikatu Sawamoto Seiki Kamisue Shougo Morichika Tomohiko Murakami Akira Yoshioka 《British journal of haematology》1995,91(3):714-721
Summary. Factor VIII (FVIII) Inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVHI binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1-5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248-2312, completely inhibited FVIII/ vWF binding in an FXISA (IC50 : 5-0 and 9-0μg/ml, respectively). Antibodies from case 3 recognizing 2170-2312 and case 5 recognizing 2170-2327 also inhibited FVIII/vWF binding (IC50 :110 and 400μg/mI, respectively). Case 4 antibodies recognizing 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti-C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308-2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti-C2 domain inhibitor alloantibodies. 相似文献
9.
In vivo neutralization of a C2 domain-specific human anti-Factor VIII inhibitor by an anti-idiotypic antibody
下载免费PDF全文
![点击此处可从《Blood》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Gilles JG Grailly SC De Maeyer M Jacquemin MG VanderElst LP Saint-Remy JM 《Blood》2004,103(7):2617-2623
Factor VIII (FVIII) administration elicits specific inhibitory antibodies (Abs) in about 25% of patients with hemophilia A. The majority of such Abs reacts with FVIII C2 domain. mAbBO2C11 is a high-affinity human monoclonal antibody (mAb) directed toward the C2 domain, which is representative of a major class of human FVIII inhibitors. Anti-idiotypic Abs were raised to mAbBO2C11 to establish their neutralizing potential toward inhibitors. One mouse anti-idiotypic mAb, mAb14C12, specifically prevented mAbBO2C11 binding to FVIII C2 domain and fully neutralized mAbBO2C11 functional inhibitory properties. Modeling of the 3-D conformation of mAb14C12 VH and alignment with the 3-D structure of the C2 domain showed putative 31 surface-exposed amino acid residues either identical or homologous to the C2 domain. These included one C2 phospholipid-binding site, Leu2251-Leu2252, but not Met2199-Phe2200. Forty putative contact residues with mAbBO2C11 were identified. mAb14C12 dose-dependently neutralized mAbBO2C11 inhibitory activity in mice with hemophilia A reconstituted with human recombinant FVIII (rFVIII), allowing full expression of FVIII activity. It also neutralized in an immunoprecipitation assay approximately 50% of polyclonal anti-C2 Abs obtained from 3 of 6 unrelated patients. mAb14C12 is the first example of an anti-idiotypic Ab that fully restores FVIII activity in vivo in the presence of an anti-C2 inhibitor. The present results establish the in vitro and in vivo proof of concept for idiotype-mediated neutralization of a major class of FVIII inhibitors. 相似文献
10.
P A Foster C A Fulcher R A Houghten S de Graaf Mahoney T S Zimmerman 《Blood coagulation & fibrinolysis》1990,1(1):9-15
The factor VIII (FVIII) binding regions of the monoclonal anti-FVIII inhibitory antibody C5 and a human FVIII inhibitor antibody have previously been reported to be contained within amino acid residues 351-365 of FVIII. Localization of the binding regions of these two antibodies was based on their reactivity with four synthetic FVIII peptides. Nineteen synthetic FVIII peptides spanning the entire acidic region of the FVIII heavy chain have now been evaluated for the ability to inhibit the binding of C5 to FVIII in an ELISA assay. The smallest peptide tested that inhibited C5 binding to FVIII consisted of residues 351-361. Those peptides that were able to inhibit C5 binding in the ELISA assay were also able to neutralize the FVIII inhibitory activity of C5 in plasma. The FVIII inhibitory activity of two human FVIII inhibitor antibodies was also partially neutralized by peptides from this region. Evaluation of the pattern of peptides reactive with the three antibodies indicates that the binding regions of these antibodies are in very close proximity to each other, but are not identical. Their respective binding regions are contained within residues 351-361 (C5), 354-362 (inhibitor 1), and 342-354 (inhibitor 2). These results suggest that this 20 amino acid segment of the acidic region of the heavy chain of FVIII may be functionally important in the expression of FVIII procoagulant activity. 相似文献
11.
Deletion of alanine 2201 in the FVIII C2 domain results in mild hemophilia A by impairing FVIII binding to VWF and phospholipids and destroys a major FVIII antigenic determinant involved in inhibitor development 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Blood》网站下载免费的PDF全文](/ch/ext_images/free.gif)
d'Oiron R Lavergne JM Lavend'homme R Benhida A Bordet JC Negrier C Peerlinck K Vermylen J Saint-Remy JM Jacquemin M 《Blood》2004,103(1):155-157
The C2 domain of factor VIII (FVIII) mediates FVIII binding to von Willebrand factor (VWF) and phospholipids (PLs), thereby determining the stability and the activity of FVIII. A deletion of Ala2201 (Del2201) was identified in the FVIII C2 domain of 2 unrelated patients with mild hemophilia A (FVIII:C 11%-33%). This mutation prevents FVIII binding to a human monoclonal antibody recognizing the C2 domain and inhibiting FVIII binding to VWF and phospholipids. By comparison to healthy FVIII, Del2201 FVIII had a significantly reduced binding to VWF, which likely contributes to reduced FVIII levels in plasma. Del2201 FVIII interaction with phospholipids was evaluated in an FXa generation assay, using various concentrations of synthetic phospholipid vesicles mimicking an activated platelet surface. At the lowest phospholipid concentration allowing FXa generation, Del2201 FVIII activity was reduced 3-fold. This is the first report of a mutation altering FVIII binding to phospholipids and occurring in patients with hemophilia A. 相似文献
12.
M. Gensana C. Altisent J. A. Aznar P. Casaña F. Hernández J. I. Jorquera M. Magallón M. Massot & L. Puig 《Haemophilia》2001,7(4):369-374
In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity. 相似文献
13.
Chin-Chin Huang Ming-Ching Shen Jen-Yang Chen Mei-Hua Hung Ting-Chang Hsu Shu-Wha Lin 《British journal of haematology》2001,113(4):915-924
Epitopes recognized by factor VIII (FVIII) inhibitors of Chinese origin were analysed by immunoblotting with full-length recombinant FVIII (rFVIII), thrombin-activated FVIII (FVIIIa) and 16 FVIII fusion proteins synthesized by bacteria. Twenty-eight patients, 12 with haemophilia A and 16 with autoimmune diseases, were recruited. Antibodies from 22 patients showed reactivity with rFVIII, 20 with FVIIIa, and one reacted only with FVIII fusion proteins. Of these 22 cases, most were reactive with A2-a2 and A3-C1-C2 of FVIII(a). Of the nine cases that depicted binding to the fusion proteins, three were reactive with the A domains, three with only the B domain, and the other three with both the A and B (or C) domains. An epitope for a neutralizing antibody of a haemophilia A patient, designated TWN-112, was localized to residues 323-390, specified by FVIII fusion proteins. The same epitope also appeared on an FVIII-expression phage library screening. Immunoabsorption of antibodies from TWN-112 with the epitope reduced the neutralizing activity of the inhibitor by 33%. The incidence of a1 of FVIII is higher, and that of a3 is lower, than previously reported. Two novel epitopes, reported for the first time in this paper, were localized on the 8B2 (amino acid residues 1022-1204) and 8A2(V) (residues 673-740) fusion proteins. These two epitopes were able to reduce inhibitory antibody activity by 24% and 25% respectively. Changes of FVIII fragment specificity were also observed in one of six patients for whom multiple samples, collected at different times, were available. Our initial finding showed that the FVIII inhibitors in these Chinese patients shared epitopes with those of patients from very different genetic backgrounds, suggesting a common mechanism for the development of FVIII inhibitors. 相似文献
14.
After treatment with factor (F) VIII concentrate a significant number of patients with hemophilia A develop inhibitory antibodies that neutralize FVIII. Epitope mapping revealed that antibodies bind to selected regions within the A2, A3, and C2 domains of FVIII. Anti-A2 and anti-A3 antibodies interfere with assembly of FVIIIa with FIXa, whereas anti-C2 antibodies impede the interaction of FVIII with phospholipids. The immunologic mechanisms underlying inhibitor development in hemophilia A have not been fully elucidated. FVIII is recognized by the immune system as a foreign antigenic substance that evokes the T cell-dependent formation of high-affinity antibodies. Clonal analysis of B cell responses in hemophilia A patients has given further insight into the epitope specificity and molecular characteristics of FVIII inhibitors. Costimulatory blockade of FVIII-reactive T cells in a mouse model for hemophilia A has suggested new approaches for treatment of inhibitor patients. In this article, recent studies on the immunobiology of FVIII inhibitors are summarized and discussed with reference to their potential impact on treatment and prevention of immune responses in patients with hemophilia. 相似文献
15.
Nogami K Shima M Nishiya K Hosokawa K Saenko EL Giddings JC Tanaka I Yoshioka A 《British journal of haematology》2002,116(4):868-874
Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti-FVIII C2 antibody (ESH8) with a Val-2248-Gly-2285 epitope inhibited FXa-catalysed FVIII activation, and that a synthetic peptide designated EP-2 (residues 2253-2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP-2 on FXa-catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP-2 inhibited FXa-catalysed activation in a clotting assay in a dose-dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP-2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose-dependent manner, but not prothrombin time. Our results indicate that EP-2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa-catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy. 相似文献
16.
Thompson AR 《Seminars in thrombosis and hemostasis》2003,29(1):11-22
Factor (F) VIII is a large gene located near the terminus of the long arm of the X chromosome. It contains 26 exons that code for a signal peptide and a 2332 amino acid polypeptide with three different types of domains, namely A1-A2-B-A3-C1-C2. The A domains are homologous with each other and those of ceruloplasmin; substitution into the known crystal structure of the copper binding protein produces molecular models. The large, central B domain is highly glycosylated but has a variable sequence, even among FVIIIs from different species. Most of B can be deleted and the resulting recombinant protein has essentially normal survival in circulation and corrects the bleeding tendency in hemophilia A patients. The C domains are similar to each other, and the crystal structure of a recombinant human C2 domain is known, allowing construction of a molecular model of C1. The FVIII protein is secreted as a heterodimer following at least two intracellular cleavages within the B domain. In circulation it is stabilized by binding to von Willebrand factor (vWF) with a plasma half-life of about 10 hours. After specific thrombin cleavages that remove the remainder of the B domain and one of the high-affinity von Willebrand factor binding sites, FVIII becomes heterotrimeric FVIIIa, capable of enhancing intrinsic FX activation by FIXa. Inactivation of FVIIIa occurs by A2 dissociation or by specific cleavages within A1 and A2 by activated protein C. Control of intrinsic FX activation is critical for hemostasis and thrombosis. 相似文献
17.
Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C-mediated proteolysis 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Blood》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nogami K Shima M Giddings JC Hosokawa K Nagata M Kamisue S Suzuki H Shibata M Saenko EL Tanaka I Yoshioka A 《Blood》2001,97(3):669-677
Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasma-derived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhydro-APC, with K(d) values of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain. The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC binding site. The findings suggest that binding of type 2 autoantibodies, recognizing residues His2009 to Val2018, protects FVIII from APC-mediated proteolysis and might contribute to the presence of FVIII immune complexes in the circulation. 相似文献
18.
A longitudinal evaluation of anti‐FVIII antibodies demonstrated IgG4 subclass is mainly correlated with high‐titre inhibitor in haemophilia A patients
下载免费PDF全文
![点击此处可从《Haemophilia》网站下载免费的PDF全文](/ch/ext_images/free.gif)
S. A. L. Montalvão A. C. Tucunduva L. H. Siqueira A. L. A. Sambo S. S. Medina M. C. Ozelo 《Haemophilia》2015,21(5):686-692
The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII‐binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti‐FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti‐FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross‐sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen–Bethesda assay. Anti‐FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma‐derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII‐binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high‐response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non‐pathologic antibody subclass with anti‐inflammatory properties in other situations, IgG4 is correlated with the presence of high‐titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII. 相似文献
19.
Computer‐predicted peptides that mimic discontinuous epitopes on the A2 domain of factor VIII
下载免费PDF全文
![点击此处可从《Haemophilia》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A. Lebreton N. Simon V. Moreau V. Demolombe C. Cayzac C. Nguyen J.‐F. Schved C. Granier G. Lavigne‐Lissalde 《Haemophilia》2015,21(3):e193-e201
Development of antibodies (Abs) against factor VIII (FVIII) is a severe complication of haemophilia A treatment. Recent publications suggest that domain specificity of anti‐FVIII antibodies, particularly during immune tolerance induction (ITI), might be related to the outcome of the treatment. Obtaining suitable tools for a fine mapping of discontinuous epitopes could thus be helpful. The aim of this study was to map discontinuous epitopes on FVIII A2 domain using a new epitope prediction functionality of the PEPOP bioinformatics tool and a peptide inhibition assay based on the Luminex technology. We predicted, selected and synthesized 40 peptides mimicking discontinuous epitopes on the A2 domain of FVIII. A new inhibition assays using Luminex technology was performed to identify peptides able to inhibit the binding of anti‐A2 Abs to A2 domain. We identified two peptides (IFKKLYHVWTKEVG and LYSRRLPKGVKHFD) able to block the binding of anti‐A2 allo‐antibodies to this domain. The three‐dimensional representation of these two peptides on the A2 domain revealed that they are localized on a limited region of A2. We also confirmed that residues 484–508 of the A2 domain define an antigenic site. We suggest that dissection of the antibody response during ITI using synthetic peptide epitopes could provide important information for the management of patients with inhibitors. 相似文献
20.
Haemophilia A: effects of inhibitory antibodies on factor VIII functional interactions and approaches to prevent their action 总被引:3,自引:0,他引:3
E. L. SAENKO N. M. ANANYEVA D. V. KOUIAVSKAIA A. V. KHRENOV J. A. M. ANDERSON M. SHIMA J. QIAN & D. SCOTT 《Haemophilia》2002,8(1):1-11
Factor VIII (FVIII) is an essential component of the intrinsic pathway of blood coagulation. Normal functioning of FVIII requires its interactions with other components of the coagulation cascade. In the circulation, it exists as a complex with von Willebrand factor (vWF). Upon activation by thrombin or activated factor X (FXa), activated FVIII (FVIIIa) functions as a cofactor for the serine protease factor IXa. Their complex assembled on the phospholipid surface activates FX to FXa, which consequently participates in formation of thrombin, the key protease of the coagulation cascade. Genetic deficiency in FVIII results in a coagulation disorder haemophilia A, which is treated by infusions of FVIII products. Approximately 25-30% of patients develop antibodies inhibiting FVIII activity (FVIII inhibitors). The major epitopes of inhibitors are located within the A2, C2 and A3 domains of the FVIII molecule. The inhibitory effects of antibodies are manifested at various stages of the FVIII functional pathway, including FVIII binding to vWF, activation of FVIII by thrombin, and FVIIIa incorporation into the Xase complex. We summarize the current knowledge of the FVIII sites involved in interaction with its physiological ligands and different classes of inhibitory antibodies and describe their inhibitory mechanisms. We outline the strategies aimed to overcome the effects of inhibitory antibodies such as development of human/porcine FVIII molecules, resistant to inhibitors. We also discuss approaches to modulate the antibody response, as well as efforts to develop a long-term immunotolerance to FVIII protein. 相似文献