High Affinity Binding of FVIII to VWF Is Not Required for the Haemostatic Effect of FVIII In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1182-1182
Author(s):  
Heidi L Holmberg ◽  
Marianne Kjalke ◽  
Ditte Karpf ◽  
Ida Hilden ◽  
Hermann Pelzer ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Clot Formation ◽  
Pharmacokinetic Studies ◽  
Affinity Binding ◽  
Maximal Effect ◽  
High Affinity Binding ◽  
High Affinity

Abstract Abstract 1182 VWF protects FVIII from clearance in the circulation and is believed to ensure location of platelets to the site of injury. However, it is unknown if binding of FVIII to VWF has a role in localizing and thereby also facilitating the effect of FVIII in vivo. In the present study, a FVIII variant, FVIII-Y1680F, lacking the high affinity binding to VWF (Leyet et al. JCB 1991; 15; 740) was used to evaluate the binding of FVIII to VWF in clot formation hemophilia A mice in vivo. Binding of the FVIII variant to immobilized VWF was evaluated by surface plasmon resonance showing a 50–25 fold reduction in the Kd for FVIII-Y1680F compared to wt FVIII. Furthermore, pharmacokinetic studies in hemophilia A mice indicated that FVIII-Y1680F is basically devoid of VWF binding in vivo. The circulating half-life decreased from 7–8 hours for wt FVIII to 0.5 hours for FVIII-Y1680F (see figure) which is comparable to the half-life of wt FVIII in VWF knockout mice (0.5 hours). Using a chromogenic assay the specific activity of FVIII-Y1680F was 9200 IU/mg similar to that of wt FVIII confirming normal activity FVIII-Y1680F after cleavage with thrombin and removal of VWF. As the short half-life may influence the haemostatic effect of FVIII-Y1680F in vivo, a 40 kDa PEG moiety was attached to the O-glycan in the B-domain of the FVIII variant. This re-establishes the circulating half-life (6.1 hours) to that of wt FVIII without affecting the specific activity in vitro. The haemostatic effect of 40K-O-PEG FVIII-Y1680F was subsequently used to investigate if high affinity VWF binding of FVIII influences its haemostatic effect in vivo. The acute haemostatic effect of 40K-O-PEG-FVIII-Y1680F was compared to wt FVIII (Advate®) in the tail bleeding model in hemophilia A mice at doses equivalent to the 50% of the maximal effect and at maximal efficacy (20 and 280 IU/kg; Elm et al., Hemophilia 2011 epub). The blood loss was significantly reduced at both doses with comparable effect of 40K-O-PEG-FVIII-Y1680F and wt FVIII (see see figure, * indicates significant differences compared to vehicle treated hemophilia A mice). This indicates that the lack of VWF binding does not interfere with the haemostatic properties of FVIII in this particular model. To further support these data, the haemostatic effect of 40K-O-PEG-FVIII-Y1680F was tested in the FeCl3 injury model (2.5, 5 and 10 IU/kg) in hemophilia A mice. No clot formation was observed in vehicle mice and 40K-O-PEG-FVIII-Y1680F normalized dose dependently the clot formation time comparable to wt FVIII. In conclusion, the current data suggest that the haemostatic effect of FVIII in vivo is not dependent on high affinity binding of FVIII to VWF. Disclosures: Holmberg: Novo Nordisk A/S: Employment. Kjalke:Novo Nordisk A/S: Employment. Karpf:Novo NOrdisk A/S: Employment. Hilden:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Koefoed-Hansen:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Thim:Novo Nordisk A/S: Employment. Karlsson:Novo Nordisk A/S: Employment. Jespersgaard:Novo Nordisk A/S: Employment. Bolt:Novo Nordisk: Employment. Stennicke:Novo Nordisk A/S: Employment.

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Peter L. Turecek ◽  
Jürgen Siekmann ◽  
Herbert Gritsch ◽  
Katalin Váradi ◽  
Rafi-Uddin Ahmad ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Full Length ◽  
Thrombin Inhibitor ◽  
Knock Out ◽  
Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

1979 ◽  
Author(s):  
B. Kudryk ◽  
M. Blombäck
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Specific Activity ◽  
High Specific Activity ◽  
Antigenic Determinants ◽  
Affinity Binding ◽  
Compact Structure ◽  
High Affinity Binding ◽  
High Affinity ◽  
Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

Towards a New Therapy for Hemophilia A: Evaluation of Multiple Approaches To Prolong the In Vivo Efficacy of Recombinant Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1606-1606
Author(s):  
John E. Murphy ◽  
Clark Pan ◽  
Baisong Mei ◽  
Jonathan Strauss ◽  
Hendri Tjandra ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Hemophilia A ◽  
Positive Impact ◽  
Active Form ◽  
Single Amino Acid ◽  
In Vitro Assays ◽  
Pharmacokinetic Studies ◽  
Disulfide Linkage

Abstract Prophylactic therapy with rFVIII has been shown to have a significant positive impact on the treatment of Hemophilia A. One of the impediments to effective prophylaxis is the requirement for frequent injections necessitated by the 12–14 hour circulating half-life of FVIII. We have evaluated a number of approaches to modify FVIII to reduce the need for frequent injections. In one approach, the active form of FVIII was stabilized by addition of a disulfide linkage between the A2 and A3 domains. As previously reported1, these molecules exhibited prolonged FVIII activity in a number of in vitro assays following activation. In vivo characterization of these molecules is in progress. Other approaches focused on increasing the circulating half-life of FVIII. In an attempt to reduce FVIII clearance by the liver receptor LRP, site-directed mutations in the reported LRP binding region of the FVIII A2 domain were generated. Twenty mutants, with single amino acid changes, were analyzed in mouse and rabbit recovery studies and no significant increase in recovery was observed. Since the point mutants may not have covered a large enough surface of the LRP binding domains, polyethlylene glygol (PEG) modification was used to disrupt a larger surface area. Previously, attachment of PEG moieties to FVIII had been shown to lead to an increase in plasma half-life in animal models; however, standard pegylation chemistries result in significant product heterogeneity and thus may not be suitable for commercial production. We have developed a novel method, based on protein engineering, to introduce PEG to specific cysteine residues on the surface of FVIII. Using this method, different molecular weight PEGs have been conjugated to sites in the A1, A2 or A3 domains of FVIII. Analysis of the pegylated proteins confirms that the attachment of PEG is highly specific to the engineered site. The pegylated products retain activity based on the two stage chromogenic assay, but exhibit reduced activity when analyzed by the one stage coagulation assay. Pharmacokinetic studies, performed in mouse and rabbit, show that pegylation increases half-life in a manner that is proportional to PEG molecular weight. Using a number of injury models in hemophilic mice, the pegylated molecules have been shown to be efficacious in stopping bleeds. The prospects for using site-specific pegylation of FVIII to produce a therapeutic for treatment of hemophilia A will be discussed.

RAT MYOMETRIAL ACTIVITY IN VIVO: EFFECTS OF OESTRADIOL-17β AND PROGESTERONE IN RELATION TO THE CONCENTRATIONS OF CYTOPLASMIC PROGESTERONE RECEPTORS

1978 ◽  
Vol 78 (1) ◽  
pp. 103-117 ◽  
Author(s):  
SANDRA J. DOWNING ◽  
S. J. LYE ◽  
JANE M. C. BRADSHAW ◽  
D. G. PORTER
Keyword(s):  
Binding Protein ◽  
Progesterone Receptors ◽  
Affinity Binding ◽  
Pregnant Rats ◽  
Pregnant Rat ◽  
High Affinity Binding ◽  
High Affinity ◽  
Repeated Doses ◽  
In Vivo Effects

The amplitude, frequency and rate of rise of intra-uterine pressure cycles in rats (postpartum, ovariectomized) were unaffected by treatment with progesterone. Amplitude was also unaffected by a combination of treatments with progesterone and oestradiol-17β, which was adequate to ensure the survival of 84% of foetuses in ovariectomized pregnant rats. The failure of progesterone to influence myometrial activity could not be attributed to a lack of 'true' progesterone receptors since these were present in the myometria of the test animals in concentrations exceeding those of oestrous animals. Evidence was obtained which suggested that a high-affinity binding protein, different from the 'true' receptor may predominate in the myometrium of the pregnant rat. Oestradiol-17β in single or repeated doses of from 0·25 to 5 μg, however, was found to reduce the frequency of pressure cycles but to increase significantly their rate of rise of pressure. There was a latency of 6–8 h in these effects of oestradiol. The possibility that inhibition of the myometrium by oestrogen may play a part in the preparation for parturition is discussed.

scholarly journals The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCADOperon Expression in Escherichia coli

2000 ◽  
Vol 182 (4) ◽  
pp. 961-966 ◽  
Author(s):  
Mireille Ansaldi ◽  
Gwénola Simon ◽  
Michèle Lepelletier ◽  
Vincent Méjean
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Response Regulator ◽  
Regulatory System ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces thetorCAD operon, which encodes the TMAO respiratory system ofEscherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torRgene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers thetorR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that thetorR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in atorS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

scholarly journals Synaptojanin cooperates in vivo with endophilin through an unexpected mechanism

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Yongming Dong ◽  
Yueyang Gou ◽  
Yi Li ◽  
Yan Liu ◽  
Jihong Bai
Keyword(s):  
Affinity Binding ◽  
Phosphatase Domain ◽  
High Affinity Binding ◽  
Bar Domain ◽  
High Affinity ◽  
Rich Domain ◽  
Phosphoinositide Phosphatase ◽  
Core Function ◽  
Membrane Bending

Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin–Amphiphysin–Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses.

scholarly journals An AU-Rich Sequence Element (UUUN[A/U]U) Downstream of the Edited C in Apolipoprotein B mRNA Is a High-Affinity Binding Site for Apobec-1: Binding of Apobec-1 to This Motif in the 3′ Untranslated Region of c-myc Increases mRNA Stability

2000 ◽  
Vol 20 (6) ◽  
pp. 1982-1992 ◽  
Author(s):  
Shrikant Anant ◽  
Nicholas O. Davidson
Keyword(s):  
Binding Site ◽  
Apolipoprotein B ◽  
Half Life ◽  
Rna Binding ◽  
Cytidine Deaminase ◽  
Binding Activity ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a Kd of ∼435 nM. A series of AU-rich templates was used to identify a high-affinity (∼50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3′ untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.

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