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Shiltagh N.,University College London | Kirkpatrick J.,University College London | Cabrita L.D.,University College London | McKinnon T.A.J.,Imperial College London | And 4 more authors.
Blood | Year: 2014

Although much of the function of von Willebrand factor (VWF) has been revealed, detailed insight into the molecular structure that enables VWF to orchestrate hemostatic processes, in particular factor VIII (FVIII) binding and stabilization in plasma, is lacking. Here, we present the high-resolution solution structure and structural dynamics of the D′ region of VWF, which constitutes the major FVIII binding site. D′ consists of 2 domains, trypsin-inhibitor-like (TIL′) and E′, of which the TIL′ domain lacks extensive secondary structure, is strikingly dynamic and harbors a cluster of pathological mutations leading to decreased FVIII binding affinity (type 2N von Willebrand disease [VWD]). This indicates that the backbone malleability of TIL′ is important for its biological activity. The principal FVIII binding site is localized to a flexible, positively charged region on TIL′, which is supported by the rigid scaffold of the TIL′ and E′ domain β sheets. Furthermore, surface-charge mapping of the TIL′E′ structure reveals a potentialmechanism for the electrostatically guided, high-affinity VWF·FVIII interaction. Our findings provide novel insights into VWF·FVIII complex formation, leading to a greater understanding of the molecular basis of the bleeding diathesis type 2N VWD. © 2014 by The American Society of Hematology. Source

Konkle B.A.,University of Washington | Stasyshyn O.,Ukrainian Academy of Sciences | Chowdary P.,Katharine Dormandy Haemophilia Center | Bevan D.H.,St. Thomas Hospital | And 8 more authors.
Blood | Year: 2015

Current management of hemophilia A includes prophylaxis with factor VIII (FVIII) replacement every 2 to 3 days. BAX 855, Baxalta's pegylated full-length recombinant FVIII (rFVIII), was designed to increase half-life and, thus, reduce the frequency of prophylactic infusions while maintaining hemostatic efficacy. BAX 855 was evaluated in previously treated patients with severe hemophilia A who were aged 12 to 65 years. A phase 1 study compared the pharmacokinetic (PK) profile of BAX 855 with that of licensed rFVIII (Advate). In a pivotal study, the annualized bleeding rate (ABR), PK parameters, and efficacy of bleeding treatment were assessed. In the phase 1 study, the mean half-life (T1/2) and the mean residence time of BAX 855 compared with Advate were 1.4- to 1.5-fold higher. These results were confirmed in the pivotal study. The pivotal study met its primary endpoint: Prophylaxis with BAX 855 resulted in an ABR that was significantly lower than half the ABR of on-demand treatment (P < .0001). The median ABR was 1.9, and 39.6% of compliant subjects had no bleeding episodes during prophylaxis, whereas subjects treated on-demand had a median ABR of 41.5. BAX 855 was also efficacious for the treatment of bleeding episodes, with 95.9% of bleeding episodes treated with 1 to 2 infusions and 96.1% having efficacy ratings of excellent/good. No FVIII inhibitory antibodies or safety signals were identified. These studies provide evidence that BAX 855 was safe and efficacious for on-demand treatment and prophylaxis administered twice weekly in patients with hemophilia A. The trials were registered at www.clinicaltrials.gov as #NCT01736475 and #NCT01599819. © 2015 by The American Society of Hematology. Source

Mannucci P.M.,University of Milan | Kempton C.,Emory University | Millar C.,Imperial College London | Romond E.,University of Kentucky | And 12 more authors.
Blood | Year: 2013

Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined ata fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF-binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post-rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞)(P < .01). Thesedata support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved. © 2013 by The American Society of Hematology. Source

Goh P.A.,University College London | Goh P.A.,National Health Service Blood and Transplant Unit | Caxaria S.,University College London | Caxaria S.,National Health Service Blood and Transplant Unit | And 8 more authors.
PLoS ONE | Year: 2013

A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells. © 2013 Goh et al. Source

Mellars G.,Katharine Dormandy Haemophilia Center | Gomez K.,Katharine Dormandy Haemophilia Center
Methods in Molecular Biology | Year: 2011

Following the discovery of the structure of DNA in 1953, it became clear that scientists needed to be able to distinguish different DNA sequences. In 1975, Edward Southern published details of a new method for detecting DNA fragments based upon their specific sequence. An indication of the importance of his work is that the technique was eponymously named after him and that subsequent methods based loosely on similar principles were named using a play on his surname (western and northern blot). The simplicity and effectiveness of the technique led to its universal acceptance as a standard method for identification of DNA sequences. In the modern laboratory where turn-around times assume ever greater importance, the process can seem relatively time-consuming. In some cases, this has led to its replacement by more rapid techniques such as long-range PCR. Nevertheless, more than 30 years after its invention, the Southern blot remains a cornerstone of molecular biology. © 2011 Humana Press. Source

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