Summary. Background: Hemophilia B is an X-linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. Objective: In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. Methods: Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo. Results: One variant containing three substitutions (V86A/E277A/R338A, FIX-Triple) exhibited 13-fold higher specific clotting activity and a 10-fold increased affinity for human FVIIIa compared with FIX-wild-type (FIX-WT) and was thus investigated systematically in vivo. Liverspecific FIX-Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5-fold higher specific clotting activity compared with FIX-WT. Human FIX-Triple and FIX-WT knock-in mice were generated and it was confirmed that FIX-Triple has 7-fold higher specific clotting activity than FIX-WT under normal physiological conditions. Protein infusion of FIX-Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX-WT. Moreover, tail-vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple in hemophilia B mice demonstrated a 7-fold higher specific clotting activity of FIX-Triple than FIX-WT. Conclusions: Our results indicate that the FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX-Triple is a good candidate for further evaluation in protein replacement therapy as well as gene-based therapeutic strategies.

We examine how a firm’s bankruptcy affects the bank financing costs of its key suppliers. We do so by using an extensive, hand-collected data set that captures the supply chain relationships of bankrupt firms over the time period 1990–2009. Looking at a sample of more than 2,000 loan contracts, we compare the average borrowing cost of suppliers in the two years prior to the bankruptcy of a key customer to the average cost in the two years following the announced bankruptcy. We find the average loan spreads increase by roughly 20% following the customer’s announced bankruptcy. These effects are even stronger if the bankrupt firm is operating within a distressed industry or when there is a strong supplier–customer relationship. We also find that the structure of lending agreements significantly changes in the aftermath of a client bankruptcy. More specifically, we find that the number of covenants increases and the lead banker(s) take an increasingly important role in the period following the client’s bankruptcy. Taken together, the results of this study provide new insights into the financial implications of supply chain changes. This paper was accepted by Uday Rajan, finance.

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Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Background Survival in hepatocellular carcinoma (HCC) is lower in the USA than in Taiwan. Little is known about the extent to which differences in stage at diagnosis and treatment contribute to this difference. We examined treatment patterns and survival in HCC and analyzed factors driving the difference. Methods Using a uniform methodology, we identified patients aged 66 years and older with newly diagnosed HCC between 2004 and 2011 in the USA and Taiwan. We compared treatment within 6 months after HCC diagnosis and 2-year stage-specific survival between the two countries. Results Compared with patients in Taiwan (n = 32,987), patients in the USA (n = 7,003) were less likely to be diagnosed as stage IA (4% vs 8%) and II (13% vs 22%), or receive cancerdirected treatments (41% vs 58%; all p < .001). Stage-specific 2-year survival rates were lower in the USA than in Taiwan (stage IA: 57% vs 77%; stage IB: 38% vs 63%; stage II: 40% vs 57%, stage III: 14% vs 18%; stage IV: 4% vs 5%, respectively; all p < .001 except p = .018 for stage IV). Differences in age and sex (combined), stage, and receipt of treatment accounted for 3.8%, 17.0%, and 16.8% of the survival difference, respectively, leaving 62.5% unexplained.