Abstract. Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 ceils. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The

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 K m 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 hemo- IntroductionFactor IX (FIX) is a vitamin K-dependent glycoprotein and an essential protease of the hemostatic system. The domain organization of FIX is shared with factors VII, X, and protein C and comprises an N-terminal domain rich in ␥-carboxyglutamic acid (Gla), 2 epidermal growth factor-like repeats and a C-terminal trypsin-like protease domain. 1 Together they form a 55-kDa single-chain protease precursor circulating in plasma at a concentration of approximately 90nM (5 g/mL), defined as 1 IU/mL. FIX is converted to the 2-chain activated form by the tissue factor (TF)-factor VIIa (FVIIa) complex or factor XIa (FXIa). Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene. The prevalence of HB is approximately 1 in 25 000 males, and it has been estimated that approximately 84 000 people are affected worldwide. 5 The mainstay in HB treatment is substitution therapy by infusion of plasma-derived or recombinant FIX (rFIX). The therapeutic goal is to prevent bleeding episodes and to provide safe and efficacious treatment of bleedings when they occur. Because of the relatively short half-life of FIX (18-24 hours [6][7][8] ), the recommended prophylaxis regimen consists of 2 to 3 weekly infusions of 40-100 IU/kg 9 FIX to maintain trough levels above 1% and thus shifting patients from a severe to a milder phenotype. When adhered to, prophylaxis in patients without severe joint disorder is efficacious with a frequency of only 0-2 breakthrough bleeds per year in the majority of patients. 8,10 However, the need for multiple weekly infusions present challen...

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