Overall, there was a 1.7 increase in the pcBF8 activity relative to phBF8, roughly equal to the increase in the pIR8 transgenic mice (Figure 1E). injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants. Introduction Hemophilia A is an X-chromosomeClinked bleeding disorder due to a deficiency in clotting factor VIII (F8), affecting approximately 1:5000 males.1,2 In this country, significant hemophilia A bleeding episodes are primarily treated by infusions of recombinant F8; however, limitations result from F8’s short half-life,3 the high cost of the replacement factor,4 and clinically relevant inhibitor development to F8 in 20%-30% of patients.5 Several studies have focused on modulating F8 hemostasis using therapeutic strategies such as the attachment of recombinant F8 to pegylated liposomes.6 Bypass products of either activated prothrombin complex concentrates or activated recombinant F8 have been successfully used in patients with inhibitors.7C9 These alternate approaches to F8 therapy do not provide continuous coverage, and may not always be effective.8,9 Gene therapy for F8 replacement is attractive as there is a wide therapeutic window for F8 corrective plasma levels.10 Past gene transfer studies have focused on liver expression of hF8; however, sustained high F8 expression levels have proven difficult to achieve in these studies.11C13 One approach to improve outcome in these studies has been to increase the efficacy of F8 by designing variants of F8 with improved in vitro activity.14 Inactivation resistant F8 (IR8) is one such variant and has increased resistance to thrombin and activated protein C inactivation in vitro.15 However, this variant also has a decreased von Willebrand factor (VWF) binding, which may limit its plasma half-life and clinical utility.16 Previous work has demonstrated that targeted delivery of ectopic hBF8 from within platelet -granules is effective at improving clotting in F8null mice.17,18 Most importantly, platelet-derived (p) human B-domainless (hB) F8 remains markedly more effective in the presence of circulating inhibitors than plasma hBF8.19C21 This pF8 is stored in -granules independent of VWF and can be effective when released at sites of injury in F8null mice.18,22 Despite having this advantage, we found that the efficacy of phBF8 in F8null mice varied based on the bleeding model studied. In particular, phBF8 was not as effective in the arteriole/venule cuticular bleeding model.17 Of greater concern was that phBF8 was associated with increased arteriole and venule embolization in the cremaster injury model. This is likely due to the observation that -granule release begins in the base of a growing clot and then extends throughout the clot, perhaps leading to inadequate fibrin generation at key U 95666E areas within the clot.23 Thus, while pF8 appears an attractive strategy for hemophilia A therapy because of its potential use in the setting of inhibitors, one also needs to carefully examine whether this nontraditional delivery system would not lead to untoward clot embolization. In this study, we tested whether using 2 F8 variants with increased F8 activity, the abovementioned IR8 and canine (c) BF8, a species of F8 that has 3- to 5-fold greater specific activity than hBF8,24,25 could decrease clot instability. We found that both F8 species were stored in murine platelets and released upon platelet activation. While pIR8 antigen reached levels comparable to phBF8, surprisingly pcBF8 was expressed at approximately 30% of the level of phBF8 using both transgenic and lentiviral expression approaches. Both of these F8s were.(A) U 95666E WT and F8null mice and (B) transgenic lines phF8.38, pIR8.5 and pcF8.24 on a F8null background were studied. Line phF8.38/F8null also showed a partial correction of platelet and fibrin accumulation, consistent with published studies.23 Initially, pIR8.5/F8null appeared not as effective at fibrin accumulation as phF8.38/F8null (Figure 3B); however, while hBF8 fibrin accumulation rapidly plateaued on the arteriole side, pIR8 fibrin curve continued to accumulate with time (supplemental Figure 2), consistent with IR8 being resistant to inactivation and showing prolonged activity in vitro. Both variants were more effective than hBF8 in cuticular bleeding and FeCl3 carotid artery models. However, in the cremaster injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants. Introduction Hemophilia A is an X-chromosomeClinked bleeding disorder due to a deficiency in clotting factor VIII (F8), affecting approximately 1:5000 males.1,2 In this country, significant hemophilia A bleeding episodes are primarily treated by infusions of recombinant F8; however, limitations result from F8’s short half-life,3 the high cost of the replacement factor,4 and clinically relevant inhibitor development to F8 in 20%-30% of patients.5 Several studies have focused on modulating F8 hemostasis using therapeutic strategies such as the attachment of recombinant F8 to pegylated liposomes.6 Bypass products of either activated prothrombin complex concentrates or activated recombinant F8 have been successfully used in patients with inhibitors.7C9 These alternate approaches to F8 therapy do not provide continuous coverage, and may not always be effective.8,9 Gene therapy for F8 replacement is attractive as there is a wide therapeutic window for F8 corrective plasma levels.10 Past gene transfer studies have focused on liver expression of hF8; however, sustained high F8 expression levels have proven difficult to achieve in these studies.11C13 One approach to improve outcome in these studies has been to increase the efficacy of F8 by designing variants of U 95666E F8 with improved in vitro activity.14 Inactivation resistant F8 (IR8) Rabbit Polyclonal to IL11RA is one such variant and has increased resistance to thrombin and activated protein C inactivation in vitro.15 However, this variant also has a decreased von Willebrand factor (VWF) binding, which may limit its plasma half-life and clinical utility.16 Previous work has demonstrated that targeted delivery of ectopic hBF8 from within platelet -granules is effective at improving clotting in F8null mice.17,18 Most importantly, platelet-derived (p) human B-domainless (hB) F8 remains markedly more effective in the presence of circulating inhibitors than plasma hBF8.19C21 This pF8 is stored in -granules independent of VWF and can be effective when released at sites of injury in F8null mice.18,22 Despite having this advantage, we found that the efficacy of phBF8 in F8null mice varied based on the bleeding model studied. In particular, phBF8 was not as effective in the arteriole/venule cuticular bleeding model.17 Of greater concern was that phBF8 was associated with increased arteriole and venule embolization in the cremaster injury model. This is likely due to the observation that -granule release begins in the base of a growing clot and then extends throughout the clot, perhaps leading to inadequate fibrin generation at key areas within the clot.23 Thus, while pF8 appears an attractive strategy for hemophilia A therapy because of its potential use in the setting of inhibitors, one also needs to carefully examine whether this nontraditional delivery system would not lead to untoward clot embolization. In this study, we tested whether using 2 F8 variants with increased F8 activity, the abovementioned IR8 and canine (c) BF8, a species of F8 that has 3- to 5-fold greater specific activity than hBF8,24,25 could decrease clot instability. We found that both F8 species were stored in murine platelets and released upon platelet activation. While pIR8 antigen reached levels comparable to phBF8, surprisingly pcBF8 was expressed at approximately 30% of the level of phBF8 using both transgenic and lentiviral expression approaches. Both of these F8s were more effective than phBF8 in the FeCl3 and cuticular model; however, only pcBF8 was more effective than phBF8 and pIR8 in the cremaster model. This improved efficacy by pcBF8 was associated with a marked decrease in embolization rate to almost that in wild-type (WT) mice. Additional observations on the efficacy of pF8 in the F8null mice were also made with regard to the benefit of concurrent low-level plasma F8 on pF8 efficacy, and on the importance of VWF binding of F8 on the protection of pF8 from circulating inhibitors. The biological and clinical implications of these findings are discussed. Methods pF8 transgenic mice construction and characterization cDNAs for IR814 and cBF826 (kindly provided by Dr Rita Sarkar and Dr Haig Kazazian, University of Pennsylvania), with Not1 restriction sites immediately upstream and downstream of the coding sequence, were.