Добірка наукової літератури з теми "Factor VIII (FVIII)"
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Статті в журналах з теми "Factor VIII (FVIII)":
1
Yada, Koji, Kenichi Ogiwara, Masaru Shibata, Midori Shima, and Keiji Nogami. "Effects of anti-factor VIII inhibitor antibodies on factor VIIa/tissue factor-catalysed activation and inactivation of factor VIII." Thrombosis and Haemostasis 105, no. 06 (2011): 989–98. http://dx.doi.org/10.1160/th10-12-0781.
Анотація:
SummaryFactor (F)VIIa/tissue factor (TF) rapidly activates FVIII activity by proteolysis at Arg372 and Arg740, and subsequently inactivates FVIIIa activity by proteolysis at Arg336, although this activation is weaker than that by thrombin. The effects of anti-FVIII inhibitor antibodies on these reactions remain unknown, however. In this study, 13 of anti-FVIII inhibitor antibodies recognising the A2 or C2 domain were prepared. None of them, irrespective of epitope specificity, significantly affected FVIIa/TFcatalysed FVIII activation in one-stage clotting assays. Anti-A2 and anti-C2 type 2 antibodies had little effect on the inactivation phase. Anti-C2 type 1 antibodies, however, modulated inactivation by 40–60% of that seen with control IgG, suggesting that the activity of FVIIIa generated by FVIIa/TF persisted in the presence of this specific type of inhibitor. SDS-PAGE analysis demonstrated that all antibodies had little effect on FVIIa/TF-catalyzed proteolysis at Arg372 and Arg740. Anti-C2 type 1, however, significantly delayed cleavage at Arg336 in dose-dependent manners. Neither anti-A2 nor anti-C2 type 2 affected this reaction, and the findings were consistent with the results of the functional assays. In addition, anti-C2 monoclonal antibodies with type 1 and 2 demonstrated similar patterns of reaction as the anti-C2 polyclonal antibodies in FVIIa/TF-mediated FVIII mechanisms. We demonstrated that FVIIa/TF activated FVIII even in the presence of anti-FVIII antibodies, but inactivation patterns appeared to depend on inhibitor type. It could be important to determine the characteristic of these inhibitor antibodies for prediction of their effects on FVIIa-related FVIII reactions, and the results could have significant therapeutic implications.Note: An account of this work was presented at the 51st annual meeting of the American Society of Hematology, 2009, New Orleans, LA, USA. This work was supported by grants for MEXT KAKENHI 21591370 in Japan and Bayer Hemophilia Award program.
2
Yada, Koji, Keiji Nogami, Kenichi Ogiwara, Katsumi Nishiya, Masahiro Takeyama, and Midori Shim. "Effects of Anti-FVIII Inhibitors On Factor VIIa/Tissue Factor-Catalyzed Activation and Inactivation of Factor VIII." Blood 114, no. 22 (November 20, 2009): 3169. http://dx.doi.org/10.1182/blood.v114.22.3169.3169.
Анотація:
Abstract Abstract 3169 Poster Board III-110 Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. We have recently demonstrated that FVIIa/TF rapidly activated FVIII by proteolysis of the heavy chain (HCh), and served physiologically as a potent activator for up-regulation of FVIII activity in very early-timed phase (ASH #1036, 2008). FVIII inhibitors develop as alloantibodies in multi-transfused patients with hemophilia A and also arise as autoantibodies in normal individuals. FVIII inactivation by inhibitors is associated with impairment of FVIII(a) cofactor function through the binding to functional crucial epitopes in FVIII. Anti-C2 inhibitors prevent FVIII binding to phospholipid, von Willebrand factor, and FXa. Anti-A2 inhibitors prevent FVIII binding to FIXa and thrombin. However, effects of these inhibitors on FVIIa action for FVIII have remained to be studied. In this study, we prepared 13 of anti-FVIII inhibitor IgGs (2 of anti-A2, 7 of anti-C2 with type 1 behavior, and 4 of anti-C2 with type 2). We first examined FVIIa/TF-catalyzed FVIII activation in the presence of anti-FVIII inhibitors in one-stage clotting assay. The levels of FVIII activity (10 nM) elevated rapidly by ∼2.0-fold within 30 sec after adding of FVIIa/TF (1 nM), and subsequently decreased to the initial level within 20 min. The presence of anti-FVIII inhibitors did not significantly affect FVIIa/TF-catalyzed FVIII activation (by 1.7∼2.2-fold) compared to normal IgG. This action was independent of the difference of inhibitor epitopes. In addition, FVIIa-catalyzed FVIIIa inactivation with anti-A2 or anti-C2 with type 2 inhibitors was little affected, similar to that with normal IgG. However, of note, all of anti-C2 with type 1 significantly inhibited FVIIa-catalyzed inactivation of FVIIIa. Inactivation rates of FVIIa with anti-C2 with type 1 (k ∼0.15) was ∼40% less than that with control IgG (k ∼0.24), supporting that the presence of anti-C2 with type 1 might persist the activity of FVIIIa generated by FVIIa. To clarify this inhibitory mechanism of anti-C2 with type 1, we performed FVIIa-catalyzed FVIII cleavage in Western blotting. FVIIa/TF (1 nM) proteolyzed the HCh of FVIII (10 nM) rapidly by cleavages at Arg372 (and Arg740), whilst cleavage at Arg336 in the A1 domain was appeared at ∼2.5 min, supporting that cleavages at Arg372 and Arg336 by FVIIa contribute to the up- and down-regulation of FVIII(a) activity, respectively. All inhibitors, independent of recognizing epitopes, did not affect FVIIa-catalyzed cleavage at Arg372. However, the presence of anti-C2 type 1 delayed the cleavage at Arg336 in timed- and dose-dependent manners, whilst either anti-A2 or anti-C2 type 2 did not affect, consistent with the functional inactivation results. FVIIa binds to the A2, A3, and C2 domains in FVIII. Based on our findings, FVIIa-interactive sites on FVIII unlikely overlapped with anti-A2 and -C2 inhibitor epitopes, and inhibition of Arg336 cleavage may be due to conformational change caused by antibody binding. Furthermore, FVIIa indeed activates FVIII even in the presence of anti-FVIII inhibitors, different from thrombin, FXa, etc, and it would be important to predict the effect of FVIIa for FVIII to determine the characteristics of anti-FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.
3
Nakajima, Yuto, Koji Yada, Keiji Nogami, and Midori Shima. "A Novel Mechanism of Factor VIIa/Tissue Factor (TF)-Catalyzed Activation and Inactivation of B-Domain-Deleted Factor VIII in the Early Initiation Phases of Coagulation." Blood 132, Supplement 1 (November 29, 2018): 1162. http://dx.doi.org/10.1182/blood-2018-99-115645.
Анотація:
Abstract We have reported that factor (F)VIII was rapidly activated by FVIIa/tissue factor (TF) in vitro by limited proteolysis of the heavy chain (HCh) at Arg372 and Arg740 in the very early-timed coagulation phase and inactivated by proteolysis at Arg336 (JTH 2010). Furthermore, the activation could be observed even in the presence of anti-FVIII inhibitors irrespective of their type of kinetics and the epitope recognized, whilst the inactivation was moderated by anti-C2 inhibitor with type 1 kinetics (Thromb Haemost 2011). A role of FVIII B-domain on FVIIa/TF-catalyzed activation and inactivation remain unknown, however. In this study, focusing on the roles of the B-domain of FVIII, we investigated the mechanism(s) of FVIIa/TF-catalyzed FVIIIa activation and inactivation by utilizing B-domain deleted (BDD)-FVIII as well as full-length (FL)-FVIII. We firstly examined FVIIa/TF-catalyzed activation and inactivation of FL- or BDD-FVIII(a) by a one-stage clotting assay. The FVIII activity (FVIII:C) of FL-FVIII (10nM) rapidly increased by ~1.7-fold within 0.5 min after addition of FVIIa (1nM)/TF (0.1nM), and subsequently decreased to the initial levels within 15 min (k = ~0.03). Interestingly, FVIII:C of BDD-FVIII (10nM), which increased up to ~1.7-fold of the initial level within 0.5 min after addition of FVIIa (1nM)/TF (0.1nM) similar to that of FL-FVIII, demonstrated a slower reduction to the initial level within 30 min (k = ~0.015) than that of FL-FVIII. In order to explore these inhibitory mechanisms of FVIIa/TF-catalyzed inactivation of BDD-FVIIIa, we investigated FVIIa/TF-catalyzed proteolytic cleavage of both BDD-FVIII and FL-FVIII by using SDS-PAGE. A rapid proteolysis in the heavy chain (Hch) of FL-FVIII within 0.5 min after addition of FVIIa/TF was observed by the cleavage at Arg740, followed by the cleavage at Arg372 and the subsequent cleavage at Arg336, consistent with our previous study. In contrast, it was of surprise that the proteolysis in the Hch of BDD-FVIII by cleavage at Arg372 was little observed after addition of FVIIa/TF, whilst that by the cleavage at Arg336 was observed within 0.5 min preceding the elevation of FVIII:C. The initial velocity of Arg336 cleavage at 0.5 min for BDD-FVIII (4.4/min) was ~3.3-times higher than that for FL-FVIII (1.4/min) by a densitometry. To the next, we examined the spontaneous dissociation of A2-domain from FVIIa/TF-catalyzed FL- or BDD-FVIIIa by a one stage clotting assay. In the presence of excess amount of A2-subunit (400nM), more than 50% of FVIIa/TF-catalyzed FVIIIa inactivation was inhibited compared to that in its absence, but no significant difference was observed between FL- and BDD-FVIII, suggesting that the spontaneous dissociation of A2-domain little affected the inhibition of the FVIIa/TF-catalyzed inactivation of BDD-FVIIIa. To further clarify the mechanism of FVIIa/TF-catalyzed BDD-FVIII activation/inactivation, we prepared and stably expressed recombinant BDD-FVIII mutants, R336A and R372A. FVIIa(1nM)/TF(0.1nM)-catalyzed activation and inactivation of R336A and R372A (10nM) was examined by a one stage clotting assay. FVIII:C of R336A and R372A rapidly increased by ~2.0-fold of the initial level within 0.5 min after addition of FVIIa/TF, similarly to that of wild type BDD-FVIII, and that of R336A subsequently decreased to the initial level within 30min (k = ~0.04), whilst little reduction of FVIII:C was observed for R372A (k = ~0.004). Evaluated by SDS-PAGE, FVIIa/TF-catalyzed proteolytic cleavage at Arg336 was predominantly observed for R372A within 0.5min after addition of FVIIa/TF, whilst cleavage at Arg372 was conversely observed for R336A. Taken together, FVIIa/TF-catalyzed activation of BDD-FVIII could be predominantly initiated by the cleavage at Arg336 or secondarily at Arg372 and resistance to the cleavage at Arg372 would hamper the subsequent inactivation. In conclusion, the B-domain of FVIII would regulate the FVIIa/TF-catalyzed activation and inactivation of FVIII by controlling the order of proteolytic cleavage at Arg336 and Arg372. We believe that our findings should also contribute to the development of more effective combination therapy of FVIIa and BDD-FVIII for hemophilia A with inhibitor. Disclosures Yada: Shire Japan Co., Ltd.: Other: Teacher at a endowed course. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.
4
Ogiwara, Kenichi, Keiji Nogami, Masahiro Okuda, Katsumi Nishiya, Masahiro Takeyama, and Midori Shima. "Interactions of Factor VIII with Tissue Factor Contributes to the Acceleration of Factor Xa Generation in the Initiation Phase of Blood Coagulation." Blood 114, no. 22 (November 20, 2009): 3177. http://dx.doi.org/10.1182/blood.v114.22.3177.3177.
Анотація:
Abstract Abstract 3177 Poster Board III-116 Activated factor (F)VII complex with tissue factor (FVIIa/TF) initiates the blood coagulation by generating FXa as extrinsic Xase complex (ex-Xase). Although FVIIa/TF also activates FIX, FIXa little functions without its cofactor, FVIIIa. A tiny amount of thrombin generated by FXa activates FV and FVIII, followed by forming of intrinsic Xase complex (in-Xase) and prothrombinase complex, respectively. These formations result in ‘thrombin burst’ and successful hemostasis. Although thrombin is thought to be a unique potent activator of FVIII in vivo, FXa and FVIIa/TF also activate FVIII in vitro. We have recently reported the detailed mechanism by which FVIIa/TF activated FVIII more rapidly in early timed-phase than thrombin (Blood Abst.1036, 2008). In this study, we further developed to examine whether TF affected FVIII(a) function. (1) FVIIa/TF rapidly increased FVIII activity by 4.7-fold of initial in the presence of Ca2+ and phospholipid (PL), following by inactivation, in one-stage clotting assay. However, since even in the presence of TF alone, FVIII activity elevated by 1.8-fold of initial, actual increase of FVIII activity by FVIIa/TF was 2.6-fold. A possibility that TF might bind to FVIIa contained in FVIII-deficient plasmas used, was negligible, since FVIIa-inhibitor used blocked an ex-Xase effect >95%. In the presence of FVIIa-inhibitor, residue FVIII activity with TF was ∼50%, thus TF alone affected FVIII cofactor activity independently of FVIIa. (2) Using SDS-PAGE, the addition of TF accelerated FVIII cleavage by FVIIa, whilst decelerated that by thrombin and FXa. (3) Surface plasmon resonance-based assays showed that FVIII(a) directly bound to TF with high affinity (Kd; ∼3 nM). (4) The effect of FVIIa/TF on in-Xase was evaluated in FXa generation assay. 0.1 nM FVIIa/TF, 1 nM FVIII, 90 nM FIX and 20 μM PL were reacted with 150 nM FX at various combinations. FVIIa/TF and FVIIa/TF/FVIII/FIX generated FXa with 3.9 and 10.4 nM/min, respectively. When FVIIa-inhibitor was added prior to addition of FX, FXa generated by FVIIa/TF and FVIIa/TF/FVIII/FIX were 5% and 46% (0.2 and 4.8 nM/min) of those without FVIIa-inhibitor, respectively. The latter was considered as FXa generated by in-Xase. Therefore, FXa derived from in-Xase was ∼40% of total FXa in this condition. (5) FVIIIa/FIXa (1 nM/2 nM)-dependent FXa generation in the presence of TF was evaluated. FXa generation in the presence of TF (0.02 and 0.3 nM) increased by ∼2 and ∼6-folds, respectively, of that in its absence. Furthermore, the functional affinity of FVIIIa for in-Xase complex in the presence of TF (0.1 nM), showed an ∼1.5-fold greater than that in its absence (Km; 4.9 ± 0.4 and 7.1 ± 0.9 nM, respectively). In conclusion, FVIIa/TF can generate FVIIIa in early timed-phase in vitro as well as FXa and FIXa, and possess potential of forming in-Xase. In addition, TF directly binds to FVIII(a), and functions in-Xase complex more efficiently by enhancing the affinity of FVIIIa for in-Xase. Although TF-dependent these reactions may be terminated rapidly via anticoagulant systems such as tissue factor pathway inhibitor, our data suggest that interactions of FVIII with TF might contribute to the acceleration of FXa generation in the initiation phase of blood coagulation. Disclosures Okuda: Sysmex Corporation: Employment.
5
Ogiwara, Kenichi, Keiji Nogami, Tetsuhiro Soeda, Tomoko Matsumoto, Katsumi Nishiya, and Midori Shima. "Mechanisms of Factor VIII Activation by Recombinant Factor VIIa Analog through Tissue Factor-Independent Manner." Blood 112, no. 11 (November 16, 2008): 1028. http://dx.doi.org/10.1182/blood.v112.11.1028.1028.
Анотація:
Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation. Analog of recombinant FVIIa (rFVIIa), NN1731 (V158D/E296V/M298Q) possesses a greater hemostatic effect than rFVIIa and has been expected in clinical application. Factor X activation rate of NN1731 compared to rFVIIa was 1.2-fold in the presence of TF (TF(+)), and was 30-fold on activated platelets in its absence (TF(−))(Allen, Arterioscler Thromb Vasc Biol.2007; 27: 683). This TF-independent mechanism likely attributes to excellent effects by NN1731. More recently, we reported the physiological role of FVIIa/TF-dependent FVIII activation in the early phase of blood coagulation. Therefore, we were tempted to investigate the action of NN1731 in FVIII activation. Time-dependent change in FVIII activity after the addition of rFVIIa/NN1731 was examined by one-stage clotting assay under the presence of phospholipids (PS:PC:PE=1:6:3), CaCl2 and TF(+)/TF(−). NN1731 raised FVIII activity up to peak level rapidly within 30 sec (TF(+)), following by inactivation. Peak level of FVIII activity by NN1731 in TF(−) reached to the same peak level of that in TF(+) within 5 min, and this peak level persisted for ~30 min. Whilst, peak FVIII level by rFVIIa in TF(−) showed only ~35% of that in TF(+) even at 30 min. FVIII activating rate of NN1731 was observed to be 1.2-fold (TF(+)) and 3.8-fold (TF(−)) of rFVIIa-catalyzed activation. Kinetics by the Xa generation assay showed the Km values of NN1731 in FVIII activation were ~1.5-fold lower than those of rFVIIa (NN1731/rFVIIa; TF(+) 27.3/49.2 nM and TF(−) 50.5/68.1 nM). Vmax values of NN1731 in FVIII activation, however, showed the obvious difference between TF(+) (2.3-fold; NN1731/rFVIIa 70.0/30.4 nM•min−1) and TF(−) (7.9-fold; 92.5/11.7 nM•min−1), compared to rFVIIa. Inactivation of FVIIIa by NN1731 was somewhat faster than that by rFVIIa. FVIII cleavages by NN1731 were analyzed using SDS-PAGE/Western blotting. The heavy chain of FVIII was proteolyzed at Arg740 (A2-B junction), Arg372 (A1-A2 junction) and Arg336 (within the A1), faster by NN1731 than by rFVIIa. These predominant cleavages by NN1731 were more evident in TF(−). However, little cleavage of the light chain of FVIII was observed by both proteases. FVIII cleavages were correlated with the observations of FVIII activation and/or inactivation. To further localize the binding region for NN1731, we evaluated the interactions between FVIII and Glu-Gly-Arg-active site modified (EGR-) NN1731, lacking enzymatic activity, in a surface plasmon resonance-based assay. The Kd value of EGR-NN1731 with FVIII was similar to that of EGR-rFVIIa (6.3 and 7.8 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains, whilst the A1 domain failed to bind, similar to the results obtained by rFVIIa. We demonstrated that NN1731 possesses higher potential as an activator for up-regulation of FVIII activity than rFVIIa. Furthermore, catalytic activity of NN1731 in TF(−), rather than binding affinity, likely attributes to this potential of its analog. We concluded that the analog has another novel mechanism in its potent hemostatic effect through FVIII activation in TF-independent manner.
6
Meeks, Shannon, Ernest T. Parker, Amy L. Dunn, John F. Healey, and Pete Lollar. "Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model." Blood 114, no. 22 (November 20, 2009): 27. http://dx.doi.org/10.1182/blood.v114.22.27.27.
Анотація:
Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.
7
Soeda, Tetsuhiro, Keiji Nogami, Tomoko Matsumoto, Kenichi Ogiwara, Katsumi Nishiya, and Midori Shima. "Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation." Blood 112, no. 11 (November 16, 2008): 1036. http://dx.doi.org/10.1182/blood.v112.11.1036.1036.
Анотація:
Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.
8
Messer, Amanda S., Barbara Ulmasov, Yogesh Kumar, Kanagasabai Vadivel, Degang Zhong, Philip Fay, and S. Paul Bajaj. "Epitope Mapping of a Monoclonal Antibody to Factor VIII That Inhibits Factor IXa:Factor VIIIa Interaction and Thrombin Activation of Factor VIII." Blood 118, no. 21 (November 18, 2011): 1177. http://dx.doi.org/10.1182/blood.v118.21.1177.1177.
Анотація:
Abstract Abstract 1177 Factor VIII (FVIII) circulates in plasma as a noncovalent heterodimer consisting of a heavy chain (HC, A1-a1-A2-a2-B domains) and a light chain (LC, a3-A3-C1-C2 domains) in a noncovalent complex with von Willebrand factor (wVF). Thrombin (IIa) cleaves FVIII between the A1-a1/A2 domains at Arg372, A2-a2/B domains at Arg740 and B-a3/A3 domains at Arg1689 generating FVIIIa that consists of an A1-a1/A2-a2/A3-C1-C2 heterotrimer. FVIIIa increases the efficiency of Factor IXa (FIXa) catalyzed activation of Factor X (FX) in a Ca2+ and phospholipid (PL) dependent manner. The A3-C1-C2 segment of FVIIIa plays an important role in FIXa:FVIIIa interaction. Here, we describe a series of experiments to map the epitope of a monoclonal antibody (mAb) that is reported to inhibit FVIII clotting activity in a one stage clotting assay (Brown et al; J Lab Clin Med, 101: 793–805, 1983). The binding of mAb to FVIII, B-domain deleted FVIII and isolated LC was assessed using surface plasmon resonance. In these experiments, mAb captured on a protein A/G coupled CM5 sensor chip served as the ligand, and FVIII and its isolated fragments served as the analytes. The Kd of binding of LC (∼40 nM) was similar to FVIII and the B-domain deleted FVIII. No binding was observed for isolated A1 and A2 domains. Further, in plasma based inhibition assays, the Kd of binding of mAb to FVIII-vWF complex and to FVIII was ∼30 nM. This suggests that the mAb epitope does not significantly overlap with the vWF binding site in the acidic a3 region of LC. Western blot analysis confirmed that the mAb is specific for the LC of FVIII. Moreover, IIa-cleaved LC starting at residue 1690 gave only a weak signal and FXa-cleaved LC starting at residue 1721 did not react with the mAb in Western blots. These data suggest that the epitope for this mAb spans the IIa-cleavage site in the LC. Consistent with these observations, the A3-C1-C2 fragment but not the C1-C2 fragment expressed in COS cells reacted with the mAb. To further define a part of the epitope in the IIa-cleaved LC, twelve A3 domain deletion fragments were constructed and expressed in E. coli. Western blot analysis of these fragments restricted the partial epitope to 1690–1710 residues of the IIa-cleaved LC. In additional experiments, the mAb did not inhibit mouse, rabbit or canine plasma FVIII in a one stage clotting assay. It did however inhibit porcine plasma FVIII with ∼40 nM Kd, sheep plasma FVIII with ∼ 68 nM Kd, and bovine plasma FVIII with ∼300 nM Kd. Analysis of the sequence alignment of residues 1680 to 1710 of FVIII from each species indicated that residues 1681 to 1694 of human FVIII most likely constitute the epitope of this mAb. The dissimilarity and the charge differences in amino acids suggest that residues Asp1681, Glu1684, Asn1685, and Ser1687 on the N terminal side and Lys1693 on the C terminal side of the IIa-cleavage site Arg1689-Ser1690 may be important for this epitope. Fluorescence energy transfer (FRET) experiments indicated that the mAb inhibits FIXa interaction with the IIa-cleaved LC consisting of A3-C1-C2 domains. In these experiments, A3-C1-C2 subunit was labeled with acrylodan (fluorescence donor) and FIXa was labeled with fluorescein-Phe-Phe-Arg-chloromethylketone (fluorescence acceptor). In the presence of FIXa, the acrylodan fluorescence was quenched indicating a biomolecular complex formation. Addition of 1.2 μM mAb abolished the acrylodan fluorescence quenching suggesting inhibition of the FIXa:LC interaction. Notably, the mAb did not inhibit activation of FX by FIXa/Ca2+/PL and FXa-cleaved FVIIIa (instead of IIa-cleaved FVIIIa). This suggests that the mAb inhibits FIXa:LC interaction by a steric hindrance and not by a direct blockage of the FIXa:LC interactive sites. In summary, the mAb inhibits clotting by preventing FVIII activation by IIa. The epitope of the mAb appears to be restricted to residues 1681–1694 of FVIII. Notably, in some of the hemophilia A patients, the epitope of the inhibitory antibodies is confined to the IIa-cleavage site including the a3 acidic domain of LC. To locate the epitope for such antibodies, one of the approaches used was to construct porcine and human FVIII hybrids. Our strategy may represent a simplified approach to locate the epitope of similar antibodies in hemophilia A patients. Such antibodies may bind strongly to LC and weakly to IIa-cleaved LC. Further, these antibodies may not bind to FXa-cleaved LC or A1/A2 subunits. Disclosures: No relevant conflicts of interest to declare.
9
Zakas, Philip, Kristopher Knight, Ernest T. Parker, H. Trent Spencer, Eric Gaucher, and Christopher B. Doering. "Bioengineering Coagulation Factor VIII through Ancestral Protein Reconstruction." Blood 126, no. 23 (December 3, 2015): 123. http://dx.doi.org/10.1182/blood.v126.23.123.123.
Анотація:
Abstract The development of transformative hemophilia A therapeutics has been hindered by the size, instability, immunogenicity and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of FVIII orthologs from existing vertebrate species, we discovered unique molecular, cellular and biochemical properties that can overcome the limitations of human FVIII. This approach facilitated the development of recombinant porcine FVIII for acquired hemophilia A and has enabled low resolution mapping and bioengineering of functional sequence determinants into human FVIII. To further extend this bioengineering approach, we employed a novel methodology termed ancestral protein reconstruction that provides certain advantages over 'rational design' approaches including a priori confidence that each ancestral FVIII is hemostatically functional. First, a mammalian FVIII phylogenetic tree with corresponding ancestral node (An) sequences was constructed through Bayesian inference using both DNA and amino acid-based models in PAML Version 4.1 (Figure 1). The limited availability of non-mammalian sequences precluded accurate ancestor prediction outside of this class. Initially, we selected 14 An-FVIII sequences for reconstruction and subsequent molecular, cellular, biochemical and immunological characterization. Each An-FVIII displayed activity in coagulation assays utilizing human hemophilia A plasma as a substrate thus demonstrating evolutionary mammalian compatibility. Infusion of highly purified preparations of several An-FVIIIs into hemophilia A mice also corrected the bleeding phenotype following a tail transection bleeding challenge confirming in vivo functionality. To study biosynthetic efficiency, secreted FVIII activity and mRNA transcript levels were analyzed following transfection of An-FVIII plasmids into HEK293 and BHK-M cell lines. An-53, common ancestor to rodents and primates, and An-68, ancestor to a subset of current rodents, displayed the highest FVIII biosynthetic efficiencies that were 12 and 15 fold greater than human FVIII, respectively (P = 0.002; Mann Whitney U test). These two An-FVIII sequences share 95 and 87% amino acid identity to human FVIII, respectively. In contrast, intermediate ancestors between An-53 and human FVIII, designated An-55, -56 and -57, do not display enhanced biosynthetic efficiency suggesting that the functional sequence determinant of high expression was lost during primate evolution. Predicting that high expression ancestral FVIIIs would be enabling to gene therapy approaches, An-53, An-68 and human FVIII cDNAs were placed in an AAV expression cassette under the control of a potent liver-specific promoter and the resulting plasmid DNA was infused hydrodynamically into hemophilia A mice. An-53 and An-68, but not human FVIII vector treated animals, achieved sustained, therapeutic plasma FVIII activity levels over 4 weeks (0.1 - 0.6 IU/ml versus <0.01 IU/ml, respectively). Recombinant An-FVIIIs were expressed, purified and biochemically characterized by SDS-PAGE, specific activity, decay following thrombin activation and inhibitor recognition. Early mammalian and all primate lineage thrombin-activated An-FVIII(a) displayed half-lives between 1.5 - 2.2 min that were not distinguishable from human FVIII. We have shown previously that modern murine, porcine, and ovine FVIIIa display significantly longer half-lives and thus this property may have evolved under positive selection. Supporting this conclusion, An-68 and An-78 display prolonged half-lives of 16 and 7 min, respectively. Lastly, the immune recognition of An-FVIIIs by a panel of A2 and C2 domain targeting inhibitory murine monoclonal antibodies as well as hemophilia A inhibitor patient plasmas was examined and many examples of reduced reactivity were revealed, which may enable the development of less immunogenic FVIII products. Herein, we report molecular discoveries that enhance our understanding of FVIII structure/function and provide a blueprint for bioengineering novel FVIII molecules with enhanced properties. These studies also show 'proof of concept' for ancestral protein reconstruction as a powerful approach to studying the biochemistry, molecular biology and evolution of the vertebrate coagulation system, which should enable identification of other new hematological drug targets and candidate biotherapeutics. Figure 1. Figure 1. Disclosures Spencer: Expression Therapeutics: Equity Ownership. Doering:Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding.
10
Radtke, Klaus-Peter, Dean Chamberlain, John H. Griffin, and Andrew J. Gale. "Whole Blood Thromboelastogram Assays Demonstrate Prolonged Factor VIIIa Potency for Recombinant Disulfide Bond-Stabilized Factor VIII Variants." Blood 104, no. 11 (November 16, 2004): 2976. http://dx.doi.org/10.1182/blood.v104.11.2976.2976.
Анотація:
Abstract Following proteolytic activation of factor VIII (FVIII) by thrombin, the FVIIIa A2 domain, A3 domain and light chain (A3-C1-C2 domains) form a non-covalent hetero-trimer. Because spontaneous A2 subunit dissociation causes loss of FVIIIa activity, we previously made two mutants each with two new Cys to form a disulfide bond linking residues 662 (A2) and 1828 (A3) (FVIIIC662-C1828) or residues 664 (A2) and 1826 (A3) (FVIIIC664-C1826). Following thrombin activation, each FVIIIa mutant was stabile compared to wild type (wt) B-domain-deleted (BDD) FVIII. Previous SDS-PAGE data showed that the A2 domain was disulfide linked to the light chain. To show that this is true for undenatured FVIIIa, here we used surface plasmon resonance (SPR) to monitor A2 dissociation from thrombin-activated wild type and variant FVIII species that were bound to the sensor surface via a monoclonal antibody. Following passage of thrombin over sensor-bound FVIII, only wt FVIII showed a characteristic decrease of SPR reflecting A2 subunit dissociation and thrombin-treated FVIIIC662-C1828 and FVIIIC664-C1826 showed only minor decreases in SPR. Thus, SPR data directly demonstrate that engineered inter-domain disulfide bridges between the A2 and A3 domains prevent A2 domain dissociation from FVIIIa. In contrast to simple plasma coagulation assays of FVIIIa, rotational thromboelastogram (RoTEG) assays of whole blood provide multiple parameters reflecting clot formation, clot quality, and clot dissolution. RoTEG assays using fresh severe hemophilia A whole blood that was reconstituted with either wt FVIII, or FVIIIC662-C1828 or FVIIIC664-C1826 were performed to test the hypothesis that the disulfide-stabilized FVIIIa mutants would show improved potency for thrombin generation. After recalcification of hemophilia A blood with added FVIII, we measured the clotting time (CT), the rate of clot-formation, the clot-firmness time (CFT), defined as the time required to reach a specified clot firmness, and the clot firmness at 5 min (CF-A5), defined as the clot firmness at 5 min after the observed CT. Samples reconstituted with disulfide-bridge-stabilized FVIII mutants or wt-FVIII had comparable CTs at similar concentrations. However, in comparison to wild type BDD-FVIII, comparable rates of clot-formation, CFTs and CF-A5 were observed for up to 10-fold lower concentrations of each disulfide-bridge-stabilized FVIII mutant. The differences between wt and FVIII mutants were especially pronounced at very low FVIII concentrations whereas at FVIII concentrations >0.01 U/mL the differences were less apparent. Because clot formation occurs early relative to overall thrombin generation which is better reflected by CFT and CF-A5 values, we interpret these data to indicate that the disulfide-stabilized FVIIIa variants provide sustained thrombin generation in whole blood compared to wt FVIII and speculate that these FVIII variants may prove superior to wt FVIII for stabilizing a hemostatic plug by providing sustained thrombin generation capacity.
Дисертації з теми "Factor VIII (FVIII)":
1
Kalandadze, Vakhtang. "Phenotypic characterization of immune cell populations in homeostatic and FVIII-challenged severe HA mice." Doctoral thesis, Università del Piemonte Orientale, 2022. http://hdl.handle.net/11579/142998.
Анотація:
Development of FVIII-neutralizing antibodies (inhibitors) is one of the main complications occurring in
30% of severe hemophilia A (HA) patients undergoing replacement therapy. Both lack of central
tolerance, due to the congenital absence of FVIII, and the presence of danger signals are factors involved
in inhibitors development, which is definitely related to the immune cells activation. Since recent studies
have been proposing new extra-coagulative roles for FVIII in the immuno-hematological biology and have
shown the presence of increased levels of several inflammatory cytokines in untreated HA patients, we
sought to investigate the effect of FVIII absence on the immune cell populations before and after
challenge with a specific (FVIII) or general (OVA) immunological challenge. We also sought to investigate
the capability of naïve CD4 T cells to differentiate into T regulatory cells. We employed a mouse model of
severe HA and we did not observe any significative change in number and percentage of primary immune
populations (e.g. T and B cells) and in the quantitative humoral response against FVIII or OVA when compare to control age- and treatment-matched animals. On the other hand, HA mice challenged with
both antigens showed higher conversion of naïve to memory T cells. Interestingly, these differences were
detected only when the immunological challenge included an adjuvant while it was absent when FVIII was
injected intravenously without any supplemental stimulus, as it happens in the normal therapeutic
regimen. Overall, the results obtained in this thesis suggest that immunogenic versus tolerogenic
response to FVIII in HA patients could depend on the reciprocal interactions of extrinsic and intrinsic
factors among which the pro-inflammatory microenvironment determined by the recurrent microbleedings
should be taken in consideration.
2
Tolley, Caroline. "Co-expression of Factor VIII with anti-FVIII Camelid antibody ligands : effect on expression levels of bio-therapeutic FVIII." Thesis, University of Kent, 2015. https://kar.kent.ac.uk/54757/.
Анотація:
Production of recombinant FVIII, the protein that is missing or dysfunctional in haemophilia A patients, is highly inefficient compared to other recombinant clotting factors such as FIX. This is predominantly due to complex intracellular trafficking, short half-life and protein instability. This study aimed to increase the amount of functional FVIII produced in mammalian cells by co-expression with anti-FVIII Camelid antibody fragments (VHH). Three VHH ligands were supplied by BAC BV (as DNA constructs), two of which when expressed in yeast are known to bind recombinant FVIII (ligands 2 and 7) and are used commercially as FVIII purification tools. From these three constructs, nine new VHH plasmids constructs were designed and transiently expressed in a stable BHK-human FVIII-expressing cell line. Of the nine VHH fragments that were co-expressed in the BHK FVIII cell line, four of these had a statistically significant impact on the ‘clotting time’ of the cell media as demonstrated by the activated partial thromboplastin time assay (aPTT). Two ligand 2 constructs (L2C1 and L2C2) prolonged the coagulation time by 4 seconds (P-value 0.0001, 95% confidence intervals 38.5-43.5), and 3.4 seconds (P-value 0.0072, 95% CI 36.5-40.3) respectively, indicating a decrease in functional FVIII activity versus media from the untransfected and null transfected BHK-FVIII cell line. Two ligand 7 constructs (L7C1 and L7C3) caused a decrease in coagulation time of 3.2 seconds (P=0.0057, 95% CI 30.5-33.3), and 4 seconds (P=0.0002, 95% CI 29.1-32.9) respectively, indicating an increase in functional FVIII activity versus media from the untransfected and null transfected BHK-FVIII cell line. Ligand 7 and ligand 2 both bind to the FVIII light chain, albeit in different regions and with different affinities (data confidential to BAC BV). BAC studies showed that ligand 7 competes with vWF on the FVIII light chain, which is known to increase stability of FVIII in vivo, whereas ligand 2 does not compete for this binding site. The opposing effects of ligand 7 and ligand 2 on FVIII clotting times seen in this study could be due to their differences in FVIII binding properties, since it is known that binding location of FVIII ligands can have an impact on FVIII clotting activity.
3
Sefiane, Thibaud. "Vers une meilleure compréhension des mécanismes moléculaires régissant l'action des thérapies non-substitutives dans l'hémophilie A et comparaison avec le facteur VIII." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASQ004.
Анотація:
L'hémophilie A est une maladie hémorragique liée à un déficit en une protéine plasmatique de la coagulation : le FVIII. Le traitement standard a longtemps consisté en un remplacement du FVIII manquant par un FVIII exogène mais l'apparition d'inhibiteurs contre le FVIII rend les thérapies substitutives inefficaces chez 30 % des patients. Récemment l'anticorps bispécifique emicizumab anti-FIX/anti-FX mimant l'action cofacteur du FVIII est arrivé sur le marché. L'objectif de ma thèse a été d'étudier comment l'emicizumab se compare au FVIII au niveau de ses propriétés moléculaires et fonctionnelles.Nous avons d'abord analysé le cas d'un patient sous prophylaxie avec emicizumab. Suite à l'apparition d'une hémarthrose après 6 mois de traitement, des anticorps anti-emicizumab ont été identifiés, entraînant une diminution de la concentration plasmatique d'emicizumab sans affecter directement son activité. Une analyse approfondie a permis de révéler le premier cas de patient traité par emicizumab avec des anticorps provoquant une clairance accélérée de l'emicizumab.Dans une seconde partie, nous nous sommes intéressés à l'impact du FVIII/FVIII-Fc vs emicizumab sur la formation et la stabilisation du caillot ainsi que sur sa structure. Des études in vitro limitées par l'absence des composants cellulaires ont été rapportées menant àdes conclusions variables. Nous avons développé un modèle murin de saignement qui a permis de montrer que l'emicizumab altère la cinétique de formation du caillot, pouvant expliquer les saignements observés chez 5% chez des patients traités par emicizumab. L'analyse microscopique de la structure des caillots indique que le FVIII et le FVIII-Fc impactent de manière similaire la structure du caillot, tandis que l'emicizumab, avec son mode d'action unique, induit des différences morphologiques significatives.Enfin, nous nous sommes intéressés à l'utilisation des modèles de saignements murins dans la détermination de l'équivalence entre les thérapies non-substitutives (emicizumab et anti-TFPI) et le FVIII. En effet, la question centrale liée à l'utilisation de ces thérapies concerne la détermination d'une éventuelle équivalence avec le FVIII. Les données issues des tests d'activités in vitro ne permettant pas de répondre à la question, nous nous sommes proposés d'évaluer in vivo cette équivalence en utilisant un panel de modèles de saignements murins. Les résultats ont permis de déterminer des équivalences variables selon la sévérité du modèle et nous ont menés à proposer qu'une équivalence absolue était illusoire et qu'il serait plus réaliste de parler d'un intervalle d'équivalence en terme d'activité FVIIIHemophilia A is a bleeding disorder linked to a deficiency in plasma coagulation protein FVIII. For a long time, the standard treatment consisted in replacing the missing FVIII with exogenous FVIII, but the emergence of inhibitors against FVIII has made replacement therapies ineffective in 30% of patients. Recently, the bispecific anti-FIX/anti-FX antibody emicizumab, which mimics the cofactor action of FVIII, was approved. The aim of my thesis was to study how emicizumab compares with FVIII in terms of its molecular and functional properties.First, we analyzed the case of a patient undergoing prophylaxis with emicizumab. After 6 months of treatment, the patient developed hemarthrosis and anti-emicizumab antibodies were identified, leading to a decrease of emicizumab plasma concentration without directly affecting its activity. Further analysis revealed the first clinical case treated by emicizumab with antibodies causing accelerated clearance of emicizumab.In a second part, we investigated the impact of FVIII/FVIII-Fc vs emicizumab on clot formation, stabilization and structure. The previously reported in vitro studies were limited by the absence of cellular components, therefore leading to contradicting results.We developed a murine bleeding model which demonstrated that emicizumab alters the kinetics of clot formation, which could explain the bleeding observed in 5% of patients treated with emicizumab. Microscopic analysis of clot structure indicates that FVIII and FVIII-Fc have a similar impact on the clot structure, while emicizumab, with its unique mode of action, induces morphological differences.Finally, we investigated the potential use of murine bleeding models to determine equivalence between non-factor therapies (emicizumab and anti-TFPI) and FVIII. Indeed, the important question linked to the use of these therapies concerns the determination of possible equivalence with FVIII. As the data from in vitro activity tests did not answer this question, we proposed to assess this equivalence in vivo using a panel of murine bleeding models. The results showed that equivalence varied according to the severity of the model and led us to propose that absolute equivalence was unrealistic. Indeed, it would be more realistic to talk about an equivalence range in terms of FVIII activity
4
Bittar, Luis Fernando 1980. "Avaliação de alterações moleculares nos genes do FVW e da ADAMTS 13 e sua correlação com os niveis plasmaticos de FVIII e FVW em pacientes com trombose venenosa profunda." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310145.
Анотація:
Orientador: Joyce Annichino-BizzacchiDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-13T01:42:43Z (GMT). No. of bitstreams: 1 Bittar_LuisFernando_M.pdf: 1309049 bytes, checksum: 8d7bbcbc4a659300d128bc58336cbb88 (MD5) Previous issue date: 2009
Resumo: Níveis elevados de fator VIII (FVIII) são um fator de risco independente e prevalente para trombose venosa profunda (TVP), e tem influência do FvW. A ADAMTS13 é responsável pela modulação do tamanho molecular do FvW, clivando os multímeros de altíssimo peso molecular. Alterações moleculares no gene da ADAMTS13 têm correlação com sua atividade. Neste estudo avaliamos a prevalência do polimorfismo A4751G no gene do FvW (região de ligação com a ADAMTS13), os polimorfismos C1797T e C1852G e a mutação C4006T no gene da ADAMTS13 em 435 pacientes com TVP (156M/279F; idade mediana=37) e 580 controles (170M/410F, idade mediana=35). Investigamos a relação entre os genótipos e a dosagem de FVIII e FvW no plasma e o risco de TVP. A dosagem de FVIII:C foi realizada por método coagulométrico de um estágio, e as dosagens de FVIII:Ag e FvW:Ag por método imunoenzimático. As alterações moleculares foram determinadas por PCR e digestão com enzimas específicas, ou SSCP e sequenciamento para confirmação. Pacientes com TVP mostraram níveis significantemente aumentados de FVIII:C (203.7 UI/dl vs. 127 UI/dl; p<0.001), FVIII:Ag (109.6 UI/dl vs. 82.4 UI/dl; p<0.001) e FvW:Ag (154.2 UI/dl vs. 108 UI/dl; p<0.001) quando comparados com o grupo controle. Não houve diferença significativa entre os grupos na prevalência das alterações moleculares estudadas. Os indivíduos com genótipo AG (FvW A4751G) apresentavam níveis significativamente reduzidos de FVIII:C (p=0.04). Embora também tenha demonstrado uma discreta associação com níveis diminuídos de FvW:Ag, esta não foi estatisticamente significativa (p= 0.07). Os indivíduos com genótipo CG (ADAMTS13 C1852G) apresentavam níveis significativamente aumentados de FVIII:Ag (p=0.05) e FvW:Ag (p= 0.01). Apesar da relação com diminuição do FVIII o polimorfismo A4751G não mostrou um efeito protetor para TVP. O polimorfismo ADAMTS13 C1852G está associado à diminuição desta metaloprotease, e sua associação com níveis aumentados de FVIII:Ag e FvW:Ag neste estudo favorece essa hipótese.
Abstract: Elevated levels of factor VIII (FVIII) are a prevalent and independent risk factor for deep venous thrombosis (DVT), and are affected by von Willebrand factor (vWF) levels. ADAMTS13 is responsible for the modulation of the molecular size of vWF, cleaving the ultra large multimers. Mutations and polymorphisms in the ADAMTS13 gene are related with its activity. This study evaluated the prevalence of polymorphism A4751G in the vWF gene, polymorphisms C1797T, C1852G and the mutation C4006T in the ADAMTS13 gene in 435 patients with DVT and 580 healthy controls. Subsequently, we investigated the relationship between the genotypes and plasma levels of FVIII and vWF and DVT risk. FVIII:C was measured by a one-stage clotting method, and FVIII:Ag and vWF:Ag were measured by chromogenic method. The molecular changes were determined by restriction endonucleases or single strand conformation polymorphism followed by sequencing. Statistical test:U Mann-Whitney, = 0.05. Patients with DVT had higher plasma levels of FVIII:C (mean 203,7 UI/dl vs. 127 UI/dl; p<0.001), FVIII:Ag (mean 109,6 UI/dl vs. 82,4 UI/dl; p<0.001) and vWF:Ag (154,2 UI/dl vs. 108 UI/dl; p<0.001) when compared to controls. We observed no statistical difference in the prevalence of all molecular changes studied between patients and controls. A4751G heterozygotes had significantly reduced levels of FVIII:C (p=0,04). Althought there was a slight association with reduced levels of vWF: Ag, this association was not statistically significant (p= 0,07). C1852G heterozygotes had significantly elevated levels of FVIII:Ag (p=0.05) and vWF:Ag (p= 0,01). Despite the relative decline of FVIII:C with the A4751G polymorphism there was no protective effect for DVT. The C1852G polymorphism is associated with a reduction of ADAMTS13, and its association with increased levels of FVIII: Ag and vWF: Ag observed in this study supports this hypothesis.
Mestrado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Mestre em Fisiopatologia Médica
5
Delignat-Heudier, Sandrine. "Stratégies thérapeutiques contre la réponse immunitaire anti-Facteur VIII chez l'hémophile A : par modification de la structure du FVIII, par inhibition de la signalisation des lymphocytes B." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066002/document.
Анотація:
L’administration de Facteur VIII thérapeutique (FVIII) chez les patients hémophiles A entraine l’apparition d’anticorps anti-FVIII appelés « inhibiteurs » chez 30% des hémophiles A sévères. Ceci constitue alors une impasse thérapeutique. Si de nombreuses investigations ont permis de caractériser les effecteurs lymphocytaires T et B impliqués dans cette réponse immunitaire, elles n’ont toutefois pas permis de proposer aux patients des stratégies thérapeutiques pour prévenir l’apparition des inhibiteurs du FVIII. La première partie de ma thèse explore la possibilité de prévenir la réponse immunitaire anti-FVIII en inhibant la capture et l’apprêtement antigénique du FVIII par les cellules présentatrices de l’antigène (CPA). Il avait été montré précédemment que les deux structures hautement mannosylées du FVIII, sur les asparagines 239 et 2118, étaient reconnues par le CD206 exprimé par les cellules dendritiques humaines dérivées de monocytes, et que cette voie d’endocytose menait à l’apprêtement antigénique du FVIII. Je me suis donc intéressée à la possibilité de réduire l’immunogénicité du FVIII en éliminant ces deux glycosylations. La seconde partie de ma thèse porte sur la possibilité de prévenir ou d’éradiquer la réponse immunitaire anti-FVIII en inhibant une molécule de la signalisation du récepteur des lymphocytes B (LB) : la tyrosine kinase de Bruton (BTK). La BTK jouant un rôle central dans la signalisation des LB, l’inhibition de celle-ci a montré un intérêt thérapeutique dans le cas de certaines pathologies malignes et auto-immunes. J’ai donc exploré le potentiel thérapeutique d’un inhibiteur de la BTK dans la réponse immunitaire anti-FVIIIAdministration of therapeutic factor VIII (FVIII) to hemophilia A patients leads to the development of anti-FVIII antibodies called “inhibitors” in 30% of severe hemophilia A patients. Despite a well characterization of T and B effectors cells involved in this immune response, there is still no therapeutic strategy proposed to the patients to prevent the occurrence of FVIII inhibitors. The first part of my thesis explores the possibility to prevent the anti-FVIII immune response by blocking FVIII capture and processing by antigen presenting cells (APC). It has been previously demonstrated that the two highly mannosylated structures on FVIII, on asparagines 239 and 2118, were recognized by the CD206 expressed on human monocyte-derived dendritic cells. This endocytic pathway led to FVIII processing and presentation to T cells. Therefore, I have investigated the possibility to reduce FVIII immunogenicity by eliminating those two glycosylations. The second part of my thesis focuses on the possibility to prevent or eradicate the anti-FVIII immune response by inhibiting a molecule involved in B cell receptor signaling: the Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cells signaling and inhibition of BTK has shown a great interest in B cell malignancies, but also in some auto-immune diseases. Therefore, I have investigated the therapeutic potential of a new BTK inhibitor against the development of the anti-FVIII immune response
6
Lavigne, Géraldine. "Caractérisation épitopique des anticorps anti-FVIII chez les patients hémophiles A traités par facteur anti-hémophilique." Montpellier 1, 2006. http://www.theses.fr/2006MON1T015.
Анотація:
"L'hémophilie A congénitale est une maladie récessive, liée au chromosome X. Elle est due à un déficit en facteur VIII (FVIII) de la coagulation. Cette maladie touche en France un enfant de sexe masculin sur 5000. L'évolution de la maladie qui à ce jour ne bénéficie que de traitements substitutifs est émaillée par des complications hémorragiques le plus souvent non extériorisées. Les plus fréquentes sont les hémorragies ostéoarticulaires en particulier les hémarthroses qui grèvent lourdement le pronostic fonctionnel. La complication iatrogène, devenue le problème majeur de la prise en charge de ces patients, est l'apparition d'anticorps dirigés contre le FVIII perfusé. Ces anticorps désignés sous le nom d'inhibiteurs surviennent chez 10 à 30% des hémophiles, qui deviennent alors résistants aux thérapies de substitution. Parallèlement à ces inhibiteurs qui interfèrent avec l'activité coagulante du FVIII plusieurs études rapportent l'existence d'anti-FVIII sans activité inhibitrice. Ceux-ci dirigés contre des épitopes dit "non fonctionnels" pourraient réduire la stabilité du FVIII ou modifier son catabolisme. La présence de tels anticorps pourrait expliquer la demi-vie réduite du FVIII transfusé observée après administration à certains patients hémophiles chez lesquels les inhibiteurs sont pourtant indétectables. Dans une première partie, nous avons produit et caractérisé des anticorps monoclonaux humains dirigés contre le domaine B du FVIII, en vue d'élucider leur mécanisme d'action. Trois d'entre eux ont été caractérisés au niveau moléculaire. Un mécanisme d'action a été proposé. L'anticorps monoclonal humain 8E3 semble interférer in vitro sur la liaison du vWF au FVIII. Nous avon ensuite étudié dans une cohorte de patients hémophiles la prévalence de ces anticorps qui n'ont pas d'action inhibitrice, et qui ne sont pas détectés par la technique Bethesda. Pour cela, nous avons opté pour une technique ELISA qui met en évidence la liaison de tous les anticorps dirigés contre le FVIII, les inhibiteurs comme les anti-FVIII "non fonctionnels". Nous avons mis au point cette technique de détection des anti-FVIII pour étudier une population de 93 patients hémophiles. Dans cette population, la prévalence des anticorps "non fonctionnels" est de 8,6% et ils sont tous dirigés contre le domaine B. Nous avons donc démontré que notre technique ELISA pouvait être intéressante, notamment dans les sous-groupes de patients sans inhibiteur, pour chercher d'autres anticorps anti-FVIII présents dans leur plasma. Dans une deuxième partie, nous avons mis au point un test rapide permettant de déterminer la répartition épitopique des anticorps anti-FVIII chez les hémophiles. En effet, si la technique ELISA est une sensible et reproductible elle est consommatrice en échantillons si l'on veut cartographier simultanément tous les épitopes cibles des anti-FVIII chez un patient. Nous avons donc mis au point une technique permettant de caractériser les épitopes cibles des anticorps allo-anticorps et auto-anticorps basée sur la technique Luminex. Cet outil présente l'avantage d'être rapide et peu consommateur de plasma. En effet, les premiers résultats nous permettent d'envisager cette technique pour caractériser les profils épitopiques des inhibiteurs de patients et de suivre leur évolution dans le temps. L'objectif de notre travail sera de valider cette technique sur une population plus importante de patients et de rechercher une relation entre le traitement utilisé (FVIII recombinant ou plasmatique) et le profil épitopique de l'inhibiteur. Cet outil pourrait alors devenir une aide précieuse comme marqueur prédictif d'efficacité d'un traitement chez un patient traité par exemple par du FVIII plasmatique contenant du facteur de Von Willebrand (spécificité épitopique pour le domaine C2 ou la région a3). "
7
Khandekar, Gauri. "Origin and Role of Factor Viia." Thesis, University of North Texas, 2013. https://digital.library.unt.edu/ark:/67531/metadc407814/.
Анотація:
Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases in zebrafish genome. On performing the genetic screen I identified 2 novel genes, hepatocytes growth factor like and prostasin involved in Factor VII activation.
8
Repessé, Yohann. "Importance du facteur von Willebrand et des mutations du FVIII pour l'allo-immunisation contre le FVIII thérapeutique chez le patient hémophile A." Caen, 2007. http://www.theses.fr/2007CAEN3086.
Анотація:
L’hémophilie A est une maladie génétique caractérisée par un déficit en FVIII, cofacteur de la coagulation plasmatique. Le traitement de l’hémophile A est substitutif et repose sur l’administration de FVIII au patient. L’apparition d’anticorps anti-FVIII « inhibiteurs » est la complication majeure du traitement. Les inhibiteurs inactivent l’activité pro-coagulante du FVIII injecté et compliquent la prise en charge des patients qui deviennent résistants aux thérapies substitutives conventionnelles. Nous nous sommes intéréssés aux facteurs de risque influençant le développement d’anticorps anti-FVIII et aux mécanismes cellulaires impliqués dans l’initiation de la réponse immunitaire anti-VIII. Dans ce travail, nous avons caractérisé les anomalies génétiques d’une population des 120 patients hémophiles A suivis au CHU de Caen. Cette étude nous a permis d’étudier la répartition des différentes mutations et d’établir des relations entre phénotypes et génotypes. Nous avons étudié la relation entre le type de mutations et le risque d’apparition d’anticorps anti-FVIII. Par ailleurs, nous avons étudié l’endocytose du FVIII par les cellules dendritiques, modèle de cellules présentatrices de l’antigène, qui correspond à l’étape la plus en amont de la réaction immunitaire anti-FVIII. Nous avons démontré que le récepteur LRP n’était pas impliqué dans ce processus et mis en évidence le rôle immunoprotecteur du VWF in vitro qui bloque l’endocytose du FVIII avec une cinétique dépendante de la dose. Ce travail permet de confirmer l’existence de facteurs de risque génétique influençant l’apparition d’inhibiteur et d’améliorer nos connaissances sur la réponse immunitaire anti-FVIIIHaemophilia A is a genetic disorder that results in insufficient levels of functional factor VIII in plasma. The treatment of bleeding episodes in hemophilia A patients involves the passive administration of exogenous human FVIII to restore normal hemostasis. The developement of anti-FVIII antibodies, called inhibitors, is the major complication of the treatment. Patients with FVIII inhibitors become resistant to conventional FVIII replacement therapy. We were interested in studying risk factors that influenced FVIII inhibitors development and cellular mechanisms involved in the initiation of the anti-FVIII immune response. In the work, we studied a population of 120 haemophilia A patients. Our results extend our insight into the mechanisms by which novel amino acid substitutions may lead to HA, and how HA patient genotypes influence the risk of FVIII inhibitor development. In addition, we studied the endocytosis of FVIII by dendritic cells, a model of professionnal antigen presenting cells, which is the first step upstream from the activation of immune effectors. Our data highlight that LRP is not implicated in FVIII endocytosis. Moreover, we described for the first time the immunoprotective effect of VWF that inhibits the endocytosis of FVIII in vitro. This work confirms the role of genetic factors in FVIII inhibitors development and improve our knowledge an the anti-FVIII immune response
9
Dahri, Latifa. "Épuration plasmatique des anticorps dirigés contre le facteur VIII de la coagulation (anti FVIII) sur des résines de polystyrène fonctionnalisé." Nancy 1, 1995. http://www.theses.fr/1995NAN10440.
10
Gilardin, Laurent. "Identification des épitopes T d’ADAMTS13 chez les patients atteints de Purpura Thrombotique Thrombocytopénique The ADAMTS13¹²³⁹-¹²⁵³ peptide is a dominant HLA-DR1-restricted CD4⁺ T-cell epitope Purpura Thrombotique Thrombocytopénique : physiopathologie, clinique, pronostic et traitement In silico calculated affinity of FVIII-derived peptides for HLA class II alleles predicts inhibitor development in haemophilia A patients with missense mutations in the F8 gene In silico prediction of immuno-dominant T-cell epitopes on human therapeutic factor VIII Predictive immunogenicity of Refacto AF Complement C3 is a novel modulator of the anti-factor VIII immune response Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS520.
Анотація:
Le purpura thrombotique thrombocytopénique (PTT) est une maladie autoimmune rare et grave caractérisée par la présence d’anticorps dirigés contre ADAMTS13 (A13), une protéase impliquée dans l’hémostase primaire. L’implication des lymphocytes T CD4⁺ spécifiques d’ADAMTS13, dans la physiopathologie de la maladie est suggérée par une restriction pour l’haplotype HLA-DRB1*11 (DR11), l’isotype IgG des anticorps. Dans ce travail, nous avons cherché à identifier les épitopes T d’A13. Tout d’abord, nous avons sélectionné in silico les peptides d’A13 susceptibles d’être présentés par les molécules HLA-DR11. Ensuite, l’étude de la liaison de ces peptides à des molécules HLA-DR11 par des tests ELISA compétitifs a permis d’identifier les peptides les plus affins. Enfin, nous avons déterminé les peptides d’ADAMTS13 reconnus par les lymphocytes T CD4⁺ de donneurs sains et de patients porteurs de l’haplotype DR11. Ces travaux ont également été reproduits pour l’haplotype HLA-DR1 et dans un modèle murin de souris transgéniques humanisées exprimant l’haplotype HLA-DR1. Les résultats de ce travail nous permettent d’envisager d’isoler des lymphocytes T CD4⁺ spécifiques d’ADAMTS13 chez les patients afin de mieux les caractériser aux différents stades de la maladie et de suivre leur évolution sous traitement dans le but de mieux anticiper les rechutesThrombotic thrombocytopenic purpura (TTP) is a rare and severe disease characterized by auto-antibodies directed against ADAMTS13 (A13), a plasmatic protein involved in haemostasis. The implication of CD4⁺ T cells in the pathogenesis of the disease is suggested by the existence of a restriction to particular HLA-DR alleles and by the IgG isotype of the antibodies. In this study, we wished to determine the T cell epitopes of A13. First, we selected in silico the immunodominant peptides, based on their binding capacity to HLA-DR11 molecules. Second, their binding capacity to purified HLA-DR11 molecules using a ELISA competitive assay led us to identify the best binder peptides. Finally, we determined the peptides recognized by human CD4⁺ T cells from DR11 healthy donors and patients. These results were reproduced for the HLA-DR1 haplotype and in a transgenic humanized HLA-DR1 mouse model. In a perspective point of view, our results will allow us to further isolate the specific CD4⁺ T cells in order to characterize them at different steps of the disease and during follow-up to better anticipate relapses
Книги з теми "Factor VIII (FVIII)":
1
Pratt, Kathleen P., and Sébastien Lacroix-Desmazes, eds. Tolerating Factor VIII: Novel Strategies to Prevent and Reverse Anti-FVIII Inhibitors. Frontiers Media SA, 2021. http://dx.doi.org/10.3389/978-2-88966-564-8.
Частини книг з теми "Factor VIII (FVIII)":
1
Herrmann, F. H., K. Wulff, G. Auerswald, J. Astermark, A. Batorova, W. Kreuz, H. Pollmann, A. Ruiz-Saez, L. Salazar-Sanchez, and S. Schulman. "Factor VII Deficiency: Clinical Manifestation and Molecular Genetics of 718 Subjects with FVII Gene Mutations." In 37th Hemophilia Symposium, 238–46. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-73535-9_51.
2
Becker, Richard C., and Frederick A. Spencer. "Fundamentals and Patient Evaluation." In Fibrinolytic and Antithrombotic Therapy. Oxford University Press, 2006. http://dx.doi.org/10.1093/oso/9780195155648.003.0033.
Анотація:
Thrombophilia is the term used to describe a tendency toward developing thrombosis. This tendency may be inherited, involving polymorphism in gene coding for platelet or clotting factor proteins, or acquired due to alterations in the constituents of blood and/or blood vessels. An inherited thrombophilia is likely if there is a history of repeated episodes of thrombosis or a family history of thromboembolism. One should also consider an inherited thrombophilia when there are no obvious predisposing factors for thrombosis or when clots occur in a patient under the age of 45. Repeated episodes of thromboembolism occurring in patients over the age of 45 raise suspicion for an occult malignancy. A summary of inherited thrombophilias are summarized in Table 24.1. This list continues to grow, as new genetic polymorphisms and combined mutations are being detected. The prevalence of common thrombophilias is shown in Figure 24.1. Factor V Leiden (FVL) mutation and hyperhomocysteinemia are present in nearly 5% of the general population and are often found in patients with venous thrombosis, while deficiencies of antithrombin (AT), protein C, and protein S are relatively uncommon. Elevated levels of factor VIII (FVIII) are uncovered frequently in the general population and in patients with thrombosis. This is not surprising as FVIII is an acute-phase reactant that increases rapidly after surgery or trauma; however, prospective studies have shown that FVIII elevation in some patients cannot be attributed to a stress reaction and probably represents mutations in the genes regulating FVIII synthesis or release (Kyrle et al., 2000). The same may be true for factors IX and XI. The relative risks for thrombosis among patients with inherited thrombophilias have been determined. While AT mutations are the least common, they are associated with a substantial risk of venous thrombosis; similar risk is seen with protein C and S deficiency. In contrast, the lifetime risk of having a thromboembolic event in an individual heterozygous for FVL is comparatively low (Martinelli et al., 1998). Incidence rates markedly increase with age, and are highest among those with AT deficiency, followed by protein C and protein S, and least with FVL.
3
Hernaningsih, Yetti. "ABO Blood Group and Thromboembolic Diseases." In Blood Groups - More Than Inheritance of Antigenic Substances [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.102757.
Анотація:
Thromboembolic diseases are usually inherited in the family. The tendency to repeat in an individual is a phenomenon that allows it to be studied. The inheritance and recurrence of thromboembolic diseases, of course, have individual risk factors for this occurrence. In the past, the ABO blood group was only needed for transfusion and organ transplant therapy. Over time, scientists think that blood type is a risk factor for certain diseases, including thromboembolism. Many studies divide between type O and non-O blood groups, both of which are distinguished by the presence of antigens on the cell surface and antibodies in the plasma of individuals. Type O does not have A, B antigens but has antibodies against A, B antigens, and vice versa for the non-O type. Many studies have shown that the non-O blood group has a risk factor for thromboembolic diseases, commonly due to higher levels of von Willebrand factor (VWF) and factor VIII (FVIII). These thromboembolic events can occur in arteries or venous. Thromboembolic manifestations are often associated with cardiovascular diseases for arterial thrombosis; and deep vein thrombosis (DVT) and pulmonary embolism (PE) for venous thromboembolism (VTE).
4
Silva, Eclesiastes Gean da, Caio Victor Barros Gonçalves da Silva, Gabriel Lúcio Guimarães dos Santos, Laís Macêdo Maciel, Bruna Barros de Queiroz, and Beatriz Santana Rocha. "Perspectivas da terapia gênica como alternativa para o tratamento da hemofilia A: uma revisão de literatura." In Avanços em Genética Humana um panorama do 1° simpósio da LAGH, 29–31. Instituto Internacional Despertando Vocações, 2024. http://dx.doi.org/10.31692/978-65-88970-44-7.29-31.
Анотація:
A hemofilia A é uma doença genética ligada ao cromossomo X, causada pela mutação no gene F8, a qual resulta numa diminuição da produção do Fator VIII da coagulação, impossibilitando que a via intrínseca da hemostasia ocorra, reduzindo significativamente a formação de coágulos. Desse modo, são necessários tratamentos vitalícios de reposição proteica do fator VIII, com objetivo de restabelecer o processo hemostático, proporcionando maior qualidade de vida aos pacientes. Porém, o medicamento possui limitações, tais como, baixo tempo de meia-vida e a indução de produção de inibidores no organismo. Neste sentido, a terapia gênica surge como um tratamento alternativo capaz de recuperar o processo homeostático através da utilização de um vetor viral mutado, induzindo a produção do Fator VIII a longo prazo. Assim, é importante conhecer a utilização da terapia gênica no tratamento da hemofilia A. Foi realizada uma busca pelos termos descritores “Hemophilia A and Factor VIII and Human Genetics and Genetic Therapy and Heredity” na base de dados PubMed. A busca resultou em 29 artigos. Como critérios de inclusão foram considerados apenas artigos na língua inglesa, publicados entre os anos de 2019 a 2024. Após a busca, foi realizada 30 a leitura dos resumos de cada um dos artigos e apenas os que abordavam diretamente os avanços do tratamento da hemofilia A por meio da terapia gênica foram selecionados, resultando em 5 artigos. A terapia gênica ocorre por meio da utilização de vírus, onde o vírus adeno-associados (AAV) pode ser utilizado1,3, Batty e colaboradores (2019) conseguiram levar a expressão do Fator VIII em pacientes após 6 semanas de uso através do AAV61 . Ohmori e colaboradores (2020), através do AAV5, induziram o aumento do fator VIII em cerca de 12 a 237%, mantendo em, no mínimo, 50% os níveis em pacientes graves para a Hemofilia A3 . O lentivírus também pode ser utilizado na terapia gênica2,4,5. Chen e colaboradores (2019), observaram que o fator de Von Willebrand e as plaquetas são os principais interferentes na terapia gênica plaquetária em camundongos com hemofilia A, apresentando imunidade anti-FVIII2 , enquanto Cai e colaboradores (2020) relataram, através do lentivírus, que é possível induzir uma tolerância imunológica específica para as plaquetas levando a uma maior eficácia protetora ao vírus, impedindo a produção de um anticorpo contra o lentivírus e assim, impossibilitando a utilização da terapia gênica4 . Gong e colaboradores (2021), por meio do gene F8-299 induziram a correção de locais de N- glicosilação com uma expressão reduzida e levando ao FVIII estável e funcional5 . Os estudos apontam para a possibilidade de o sistema imune levar à produção de anticorpos contra os vírus utilizados durante a terapia mesmo com riscos mínimos. Observa-se que a terapia gênica possui um potencial no tratamento da hemofilia A, uma vez que é possível trazer a expressão do Fator VIII de forma ativa por tempo prolongado, tornando menor a necessidade do tratamento de reposição proteica. Portanto, requerem mais estudos clínicos em humanos a respeito da terapia gênica na hemofilia A.
5
Becker, Richard C., and Frederick A. Spencer. "Historical Perspectives in Hemostasis, Coagulation, and Fibrinolysis: A Foundation for Understanding Thrombotic Disorders and Developing Effective Treatment." In Fibrinolytic and Antithrombotic Therapy. Oxford University Press, 2006. http://dx.doi.org/10.1093/oso/9780195155648.003.0005.
Анотація:
Hemostasis, the prompt cessation of bleeding at a site of vascular injury, is among the most fundamental physiologic and teleologically vital defense mechanisms in nature. Without a functionally intact hemostatic mechanism, death could ensue rapidly even after minor traumas associated with everyday life. In mammalian blood coagulation, regulated by a complex network of integrated biochemical events, five protease factors (f ) (fIIa [thrombin], fVIIa, fIXa, fXa, and protein C) interact with five cofactors (tissue factor, f VIIIa, fVa, thrombomodulin, and protein S) to regulate the generation of fibrin (Davidson et al., 2003). Although each component of the mammalian coagulation network has unique functional properties, available data based on gene organizations, protein structure, and sequence analysis suggest that it may have resulted from the reduplication and diversification of two gene structures over 400 million years ago. A vitamin K–dependent serine protease is composed of a γ-carboxylated glutamic acid (GLA) epidermal growth factor-like (EGF) 1–EGF 2-serine protease domain structure common to fVII, fIX, fX, and protein C, and the A1-A2-B-AB-C1-C2 domain structure common to fV and fVIII. Prothrombin is also a vitamin K–dependent serine protease; however, it contains kringle domains rather than EGF domains (suggesting a replacement during gene duplication and shuffling). Analyses of active site function amino acid residues reveal distinguishing characteristics of thrombin from other serine proteases, supporting its position as the ancestral blood enzyme (Krem and Cera, 2002; McLysaght et al., 2002). The rapid transformation of fluid blood to a gel-like substance (clot) has been a topic of great interest to scientists, physicians, and philosophers since the days of Plato and Aristotle ( Jewett, 1892; Lee, 1952). However, it was not until the beginning of the 18th century that blood clotting (coagulation) was appreciated as a means to stem blood loss from wounds (hemostasis) (Petit, 1731). As with other areas of science, the microscope played a pivotal role in the understanding of coagulation. In the mid-17th century, Marcello Malpighi separated the individual components of a blood clot into fibers, cells, and serum (Forester, 1956).
Тези доповідей конференцій з теми "Factor VIII (FVIII)":
1
Koedam, Joost A., Rob J. Hamer, Nel H. Beeser-Visser, Etienne Jap Tjoen San, Kees Schippers, and Jan J. Sixma. "THE INTERACTION BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644771.
Анотація:
Factor VIII (FVIII) circulates in plasma as a non-covalent complex with von Willebrand factor (VWF), a large multimeric adhesive glycoprotein. VWF serves as a carrier for FVIII and is thought to stabilize FVIII. The interaction between the two proteins was studied by binding purified human 125I-FVIII to VWF which was coated on a solid matrix. Experiments employing isolated heavy and light chains of FVIII and monoclonal antibodies indicated that binding occurred through the carboxyterminal 80kDa light chain of factor VIII. Treatment of VWF-bound 125I-FVIII with thrombin resulted in the release of a light chain-derived 70kDa fragment and a heavy chain-derived 50kDa fragment. A 42kDa heavy chain-derived fragment was found in the fraction which remained bound to VWF. Treatment with factor Xa (FXa) resulted in the release of 63, 50, 45, and 42kDa fragments. No phospholipids were required for proteolysis of FVIII by either of these enzymes. In solution, the activation of FVIII by FXa, but not by thrombin, was inhibited by VWF. Neither activation, nor cleavage or release from VWF were observed when FVIII was incubated with factor IXa. Activation of FVIII was parallelled by its release from VWF. We conclude that the thrombin-activated form of FVIII consists of a complex between the 70kDa and 50kDa fragments. Inactivation of FVIII by activated protein C (APC) was inhibited when FVIII was complexed to VWF. This protective effect of VWF was abolished upon activation of FVIII and its subsequent release from VWF.In order to locate the binding site for FVIII on the VWF molecule, we digested VWF with Staphylococcal V8 protease (Sp). Digestion products were isolated with Mono Q ion-exchange chromatography and identified as Spl (39 kDa), SpII dimers (220 kDa) and Spill dimers (a triplet ranging from 210-280 kDa) by their molecular weight and chromatographic behaviour (J.-P. Girma et al.. Biochemistry 1986, 25:3156-3163). Purified VWF or digestion products were spotted on nitrocellulose paper, followed by blocking with an albumin solution. Binding of FVIII was studied by incubating the filters with 125I-FVIII, followed by autoradiography. Fifty ng of VWF was sufficient in order to detect FVIII binding. No binding was observed to partially reduced dimeric undigested VWF. Of the isolated digestion products, only the SpIII dimer was able to bind 125I-FVIII. After Western blotting of VWF-fragments from SDS-polyacrylamide gels, 125I-FVIII bound only to the bands which represented SpIII. Therefore, the domain on VWF responsible for the binding of FVIII seems to be located on its aminoterminal SpIII fragment. The integrity of internal disulfide bonds and dimerisation of VWF are required for FVIII binding.
2
Fulcher, C. A., R. A. Houghten, S. de Graaf Mahoney, J. R. Roberts, and T. S. Zimmerman. "SYNTHETIC PEPTIDE PROBES OF FACTOR VIII IMMUNOLOGY AND FUNCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644768.
Анотація:
In order to develop specific immunologic reagents for mapping functionally important sites on FVIII, we have prepared rabbit polyclonal antibodies against synthetic peptides of FVIII derived from regions along the entire FVIII amino acid sequence. To date, a total of 70 peptides have been synthesized and characterized by amino acid and HPLC analysis. The peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as a linkage reagent and used to immunize rabbits. Antisera were tested by ELISA assay on polystyrene microtiter plates coated with either the peptide immunogen, or purified FVIII. The antisera were also tested for their ability to inhibit FVIII clotting activity and to react with separated FVIII polypeptides on immunoblots.Of the 70 peptides, all reacted with the peptide immunogen, 45 reacted with purified FVIII and 33 reacted with FVIII on immunoblots. Because we had obtained evidence that cleavage of the amino terminal region of the 80 kDa polypeptide may play a role in FVIII activation by thrombin, a series of partially overlapping peptides, 15 residues in length, were synthesized in this area. After affinity purifying these antibodies on columns of FVIII immobilized on agarose, adjusting the antibodies to equal antigen binding titers by dot immunoblotting and testing for inhibition of FVIII activity, only one antibody could strongly inhibit FVIII clotting activity. This inhibition could be blocked by the peptide itself at nanomolar concentrations and no significant inhibition could be shown by antibodies to partially overlapping peptides individually, or in combination. These data suggest that a site important to FVIII function can be localized to a 15 amino acid residue region of the 80 kDa polypeptide of FVIII. In addition, a second inhibitoryantibody was identified which was produced against a peptide in the carboxy terminal region of the 54 kDa thrombin fragment of FVIII and this area is currently being studied in a similar manner. In addition, two monoclonal anti-FVIII synthetic peptide antibodies have been produced which react with purified FVIII on immunoblots. One of these antibodies also functions as an immunoadsorbent when linked to agarose and FVII can be purified in this manner, using the synthetic peptide as eluant. It is evident that antibodies to synthetic peptides of FVIII can be useful probes of FVIII structure, function and interactions as well as being of use in FVIII purification.
3
Tran, T. H., U. Zuhlke, J. Hauert, F. Duckert, G. A. Marbet, and R. Wagenwoord. "INFLUENCE OF HEPARIN ON FACTOR VIII (FVIII) ASSAY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644031.
Анотація:
Thrombin-activated FVIII accelerates the conversion of factor X to activated factor X (FXa) by activated factor IX, phospholipids and calcium ions. In plasma, FVIII is activated by initial traces of thrombin, which, in the presence of heparin, is rapidly inhibited by binding to anti thrombin III and heparin cofactor II. To avoid the effect, we have experienced with increasing amounts of exogenous thrombin. We were able to match the heparin cofactors concentration in diluted plasma with thrombin, so that the presence of heparin did not affect the formation of FXa, whose activity was assessed with a chromogenic substrate. Indeed, addition of heparin at any concentration to citrated plasma showed no significant deviation from the FVIII control value. Levels of FVIII in heparinized plasmas similar to those in citrated plasmas further confirmed the finding. Patients plasmas showed comparable FVIII levels before and after heparin infusion, though plasma PTT was clearly prolonged after in vivo heparinization. FVIII chromogenic assay was correlated with the one-stage clotting assay by measuring FVIII levels in 60 hemophiliacs A and carriers, in patients with von Willebrand disease (27) and other congenital deficiencies (4), high risk of thrombosis (15), bleeding tendency (20), disseminated intravascular coagulation (4) and circulating anticoagulants (2), and commercial concentrates. There was a highly significant correlation between both techniques (N=127, r=0.97, Y=0.91X + 4, range 1-380%). Three severe hemophiliacs with were detected with both methods. Data obtained from both techniques were also in good agreement in the range of 1-20% FVIII.Thrombin was added both to activate instantaneously FVIII and to neutralize heparin cofactors in samples. It thus abolishes the incubation time needed to generate in situ traces of thrombin and the influence of heparin on our FVIII assay. An eventual fibrin formation does not affect the FXa formation and the reading. The technique is also suited for automation.
4
Patrassi, G. M., A. Santarossa, F. Fallo, M. T. Sartori, M. Viero, and A. Girolami. "FACTOR VIII AND FACTOR XII LEVELS IN BORDERLINE HYPERTENSION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644259.
Анотація:
Borderline hypertension causes mortality and morbidity rates similar to those associated with estabilished hypertension. However, there is no univocal guideline for its therapeutic management. Hypercoagulability in hypertension has been demonstrated. The aim of our study was to evaluate some coagulation factors in agroup of patients affected by borderlinehypertension. The following tests were carried out: PT and PTT, Factor VIII coagulant activity, FVIII antigen and FVIII ristocetin cofactor, Factor XII and Factor XI activities. These tests were selected for their relationship to the contact coagulative activation near the vascular wall. In our patients statistically significant higher FVIII and FXII coagulant activities than normal control subjects were found. Moreover, an evident even though not statistically significant PTT shortening was seen. Other tests taken into consideration were all within normal limits. Our results suggest that an increased FVIII and FXII synthesis and/or release is present, and an activated coagulation system exists in borderline hypertension. Furthermore, it is not clear why an excess of FVIII:C over FVIIIR:Ag and FVIIIR:RCof was found in our patients. In conclusion, an activation of haemostatic mechanism was found in borderline hypertension. The young age of patients and the absence of evident hypertensive angiopathy are in agreement with an overactivity of blood vessel tone. Haemostatic activation could be an useful marker in favour of the precious management of patients with borderline hypertension.
5
Joost, S., A. Koedam, Joost C. M. Meijers, Jan J. Sixma, and Bonno N. Bouma. "VON WILLEBRAND FACTOR PROTECTS FACTOR VIII FROM INACTIVATION BY ACTIVATED PROTEIN C AND PROTEIN S." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643618.
Анотація:
Activated protein C (APC) inactivated the cofactors factor V (FV) and factor VIII (FVIII). In the case of FV, this reaction and the respective roles of Ca2+ , phospholipids and protein S have been well documented. We investigated the role of protein S and von Willebrand factor (VWF) on the inactivation of FVIII.Purified human factor VIII (3 units/ml) was incubated with protein C (0.2 μg/ml) in the presence of 8 μg/ml phospholipid, 5 mM CaCl, and 1 unit/ml hirudin. Factor VIII coagulant activity decreased with a pseudo first-order rate constant of 0.09 min . The reaction rate increased linearly with the concentration of prot^ig S in the incubation mixture. 12I-FVIII was incubated under the same conditions. SDS-polyacrylamide gel electrophoresis showed cleavage products of Mr 43 and 22 kDa. High Mr bands (FVIII-heavy chain) ranging fromMr 108 to208 kDa disappeared while the Mr 80 kDa FVIII-lightchain remained unchanged. The degradationpattern was not changed by addition of protein S.The FVIII-VWF complex was reconstitutedby mixing the two components (±2 units VWF/units FVIII) and lowering the calcium concentration to 2 mM. The inactivation of the FVIII-VWF complex by APC proceeded at a 15- to 20-fold slower rate as compared to the isolated FVIII, indicating a protection of FVIII by VWF. Protein S exhibited no cofactor activity on the inactivation of FVIII-VWF by APC. The protective effect of VWF was lost completely after activation of the FVIII-VWF complexwith thrombin (0.05 units/ml).When FVIII (0.1 units/ml) was added toplasma of a patient with severe von Willebrand's disease, 96% of its activitywas lost in 20 min after the addition of APC. All of the FVIII activity was retained when haemophilic plasma was used. Mixing experiments showed that one unit ofVWF unit FVIII is needed to fully protec FVIII against APC. These results may explain the observed lability of FVIII in von Willebrand's disease patients.
6
Grant, P. J., K. K. Hampton, P. G. Wiles, and C. R. M. Prentice. "THE EFFECTS OF VASOPRESSIN ON FIBRINOLYSIS AND FACTOR VIII ARE NOT MEDIATED THROUGH V2 RECEPTORS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644712.
Анотація:
Vasopressin (aVP) mediates its effects on smooth muscle through V1 receptors and on the kidney via pharmacologically distinct V2 receptors. Infusions of aVP and its long acting synthetic analogue DDAVP both produce increases in factor VIII and fibrinolytic activity in man. V1 receptors are known not to mediate this effect, however it has been suggested that the FVIII response might be mediated by V2 receptors as patients with nephrogenic diabetes insipidus are reported to have no FVIII response to DDAVP. It remains unclear whether this is a true phenomenon or reflects tachyphylaxis to the high vasopressin levels found in nephrogenic diabetes insipidus. The aim of this study was to investigate whether the pharmacological V2 receptor blocker lithium alters the effect of aVP infusions on FVIII and fibrinolysis in man. 4 control subjects and 6 patients taking long term lithium therapy (mean serum lithium 1.09 mmbl/l) were infused with 2.0 units aVP over 1 hour. Samples were collected for assay of aVP, euglobulin clot lysis time (ECLT) and FVIII coagulant activity (FVIIIC) before and at the end of infusion. In the control subjects median aVP rose from 0.5 to 83 pg/ml at the end of infusion. FVIIIC rose frcm 100 to 333% and plasminogen activator activity (PAA: 106 /ECLT) from 198 to 437 units. In the lithium treated group median aVP rose frcm 0.5 to 68 pg/ml at the end of infusion. FVIIIC rose from 100 to 263% and PAA from 102 to 453 units. There was a significant correlation between the plasma aVP and FVIIIC (r = 0.89 p < 0.005) and PAA (r = 0.92 p < 0.001) in the control group and the lithium treated group (FVIIIC r = 0.81 p < 0.002; PAA r = 0.69 p < 0.02). There was no significant difference between the rise in either FVIIIC or PAA in the lithium treated group compared with controls. These results do not support the hypothesis that the action of aVP on FVIII or fibrinolysis is mediated by V2 receptors. The effects of aVP on haemostasis may either be mediated directly through a third class of receptor or indirectly by the release of an intermediate hormone.
7
Váradi, K., J. Kárpáti, and S. Elödi. "ENZYME LINKED IMMUNOASSAY (ELISA) FOR FACTOR VIII ANTIGEN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644033.
Анотація:
A two site ELISA test was developed for measuring factor VIII antigen (FVIIIsAg). The assay is based on two antibodies developed in a non-haemophilic and in a severe haemophilia-A patient, respectively. The IgG fraction prepared from the non-haemophilic plasma was used for coating, and the IgG isolated from the haemophilia-A plasma was labelled with horse-radish peroxidaseFVIII:Ag and FVIII activity was measured in 28 healthy blood donors and in 41 haemophilia-A patients. The normal range for FVIIIcAg was 40 - 180 %, the correlation coefficient between FVIII:Ag and FVIII activity assays was 0.8. The sensitivity of the assay ranges between 0.005 - 0.2 U/ml FVIII:Ag. In 18 cases of severe haemophilia-A FVIII:Ag was not detectable. In 3 out of 23 mild haemophilia-A cases FVIIIrAg was significantly higher, then FVIII activity, indicating CRM variants of the disease. Due to the high sensitivity of FVIII:Ag detection, the assay appears to be suitable for prenatal diagnosis
8
Pötzsch, B., U. Delvos, E. Anders, N. Heimburger, and G. Müller-Berghaus. "FACTOR VIII/VON WILLEBRAND FACTOR COMPLEX: ONLY THE 440 000 SUBUNIT OF ENDOTHELIAL CELL-DERIVED VON WILLEBRAND FACTOR FORMS A COMPLEX WITH PURIFIED PLASMA FACTOR VIIIC." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644092.
Анотація:
Von Willebrand Factor (vWF) circulates in plasma as a series of multimers with moleculag weight ranging from M = 0.44 x 106 up to more than 20 x 106 . Besides the mediation of platelet adhesion to exposed subendothelium, the protein plays an important role in the stabilization and the transport of Factor VIIIC (FVIIIC). In the present study the interaction between FVIIIC and vWF was studied by recombination experiments. vWF was isolated from cultured human umbilical vein endothelial cells by immunoprécipitation. This source of vWF ascertained, that it was free of FVIIIC as indicated by the absence of FVIIIC activity as well as FVIIIC antigen. FVIIIC was prepared by immunoabsorption from human plasma yielding an activity of 1600 IJ/mg. SDS-PAGE analysis showed two main bands at Mr= 0.28 x 106 and 0.18 x 106 , respectively. vWF-multimers were separated by SDS agarose gel electrophoresis and were electrophoretically transferred onto nitrocellulose sheets. After extensive washing, the sheets were incubated for 12 h with 20 U/ml FVIIIC in PBS, pH 7.4, containing 2.5 mM calcium chloride. Subsequently, associated FVIIIC was detected by autoradiography with a 125-I-labelled monoclonal mouse anti-(human FVIIIC) antibody. The results of recombination experiments exclusively showed prominent staining of the Mr= 0.44 x 106 vWF band in the autoradiography. However, proteolytically degraded FVIIIC with partly retained procoagulant activity did not show a positive stain. The results indicate that an intact FVIIIC molecule and the smallest multimer of vWF are required for the formation of a stable FVIII/vWF complex.
9
Pittman, Debra D., Louise C. Wasley, Beth L. Murray, Jack H. Wang, and Randal J. Kaufman. "ANALYSIS OF STRUCTURAL REQUIREMENTS FOR FACTOR VIII FUNCTION USING SITE-DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644044.
Анотація:
Factor VIII (fVIII) functions in the intrinsic pathway of coagulation as the cofactor for Factor IXa proteolytic activation of Factor X. fVIII contains multiple sites which are susceptible to cleavage by thrombin, Factor Xa, and activate) protein C. Proteolytic cleavage is required for cofactor activity and may be responsible for inactivation of cofactor activity. In order to identify the role ofthe individual cleavages of fVIII in its activation and inactivation, site-directed DNA mediated mutagenesis of fVIII was performed and the altered forms of fVIII produced and characterized. Conversionof Arg residues to lie residues at amino acid positions 740, 1648, and 1721 resulted in resistance to thrombin cleavage at those siteswith no alteration of in vitro procoagulant activity. Modification of the thrombin cleavage sites at either positions 372 or 1689 resulted in loss of cofactor activity suggesting that these sites are important for activation. Modification of the postulated activated protein C cleavage site at position 336 resulted in fVIII with a higher specific activity than wild type, possibly due to resistance toproteolytic inactivation.DNA mediated mutagenesis was also used to study the role of post-translational biosynthetic modifications of fVIII. Structural characterization of recombinant fVIII suggested the presence of sulfated tyrosine residues within two acidic regions located between amino acid residues 336-372 and 1648-1689. Individual modification of theseTyr residues to Phe had negligible effect on synthesis and in vitrocofactor activity. The effect of combinations of these mutations onsecretion, cofactor activity, and vWF interaction will be presented.
10
Verbeet, M. Ph, R. F. Evers, A. Leyte, H. L. Lamain, A. J. J. Van Ooyen, J. A. Van Mourik, and H. Pannekoek. "DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643613.
Анотація:
Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.