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- Ostergaard, Henrik, et al.
(författare)
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Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide
- 2011
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 118:8, s. 2333-2341
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Tidskriftsartikel (refereegranskat)abstract
- Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the Km for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency. (Blood. 2011; 118(8): 2333-2341)
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| 2. |
- Siegbahn, Agneta, et al.
(författare)
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Regulation of chemotaxis by the cytoplasmic domain of tissue factor
- 2005
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Ingår i: Thrombosis and Haemostasis. - 0340-6245 .- 2567-689X. ; 93:1, s. 27-34
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Tidskriftsartikel (refereegranskat)abstract
- We previously demonstrated that FVIIa bound to tissue factor (TF) induces a hyperchemotactic response towards PDGF-BB. The aim of the present study was to investigate the role of the cytoplasmic domain of TF in cell migration. Porcine aortic endothelial (PAE) cells expressing human PDGF beta-receptors (PAE/PDGFRbeta) were transfected for expression of human wildtype TF (PAE/PDGFRbeta,TF), a construct lacking the cytoplasmic domain (PAE/PDGFRbeta,TFDeltacyto), a construct with alanine replacement of serine 258 (PAE/PDGFRbeta,TFS258A), or a construct with alanine replacement of serine 253, 258 and 263 in the cytoplasmic domain (PAE/PDGFRbeta,TF3SA). All stably transfected cell lines expressed functional TF. Chemotaxis was analyzed in a modified Boyden chamber assay. PAE/PDGFRbeta,TF cells stimulated with FVIIa migrated towards a 100-fold lower concentration of PDGF-BB than in the absence of FVIIa, however, hyperchemotaxis was not seen in PAE/PDGFRbeta,TFDeltacyto cells. PAE/PDGFRbeta/TFS258A and PAE/PDGFRbeta,TF3SA cells responded to low levels of PDGF-BB, but migrated a significantly shorter distance than PAE/PDGFRbeta,TF cells. Thus, hyperchemotaxis towards PDGF-BB is likely to depend in part on phosphorylation of the cytoplasmic domain of TF. We conclude that the cytoplasmic domain of TF plays a pivotal role in modulating cellular migration response. Our results suggest that the FVIIa/TF complex mediates intracellular signaling by alternative signal transduction pathway(s).
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