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Concept: Factor VII

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During cesarean delivery in patients with placenta previa, hemorrhaging after removal of the placenta is often challenging. In this condition, the extraordinarily high concentration of tissue factor at the placenta site may constitute a principal of treatment as it activates coagulation very effectively. The presumption, however, is that tissue factor is bound to activated factor VII (FVIIa).

Concepts: Childbirth, Obstetrics, Caesarean section, Placental abruption, Placenta accreta, Placenta praevia, Factor VII

33

Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.

Concepts: Blood, Coagulation, Haemophilia A, Haemophilia B, Factor X, Factor XI, Factor VII, Factor IX

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A global Phase 3 study evaluated the pharmacokinetics, efficacy and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (FIX activity ≤ 2%). The study included 2 groups: Group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10- or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; Group 2 patients received on-demand treatment for bleeding episodes for 26 weeks and then switched to a 7 day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous factor IX (FIX) treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was a 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P <0.0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. Clinicaltrials.gov (NCT0101496274).

Concepts: Blood, Coagulation, Haemophilia B, Factor X, Factor VII, Factor IX

27

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study.

Concepts: Pharmacology, Understanding, Effectiveness, Distribution, Pharmacokinetics, Factor VII

26

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for post-translational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multi-component nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a HEK 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of PRGP2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites; observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8 positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo.

Concepts: Protein, Cell, Coagulation, Posttranslational modification, Warfarin, Vitamin K, Factor X, Factor VII

25

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18) F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18) F]fluorobenzoate, and the [(18) F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18) F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18) F]ASIS and ASIS could be detected. Furthermore, [(18) F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18) F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18) F]ASIS are in progress.

Concepts: Protein, Cancer, Oncology, Positron emission tomography, Positron, In vivo, In vitro, Factor VII

25

Case reports on recombinant human factor VIIa (rhuFVIIa) use in women with severe postpartum hemorrhage (PPH) showed encouraging results, but no randomized controlled trial (RCT) is available.

Concepts: Childbirth, Randomized controlled trial, Factor VII

22

Factor Xa (FXa) is known to play a central role in a blood coagulation cascade and considered to be one of the most attractive targets for oral anticoagulants of new generation.

Concepts: Coagulation, Warfarin, Heparin, Vitamin K, Factor X, Factor VII, Tenase

15

The autosomal dominantly inherited east Texas bleeding disorder is linked to an A2440G variant in exon 13 of the F5 gene. Affected individuals have normal levels of coagulation factor V (FV) activity, but demonstrate inhibition of global coagulation tests. We demonstrated that the A2440G mutation causes upregulation of an alternatively spliced F5 transcript that results in an in-frame deletion of 702 amino acids of the large activation fragment, the B domain. The approximately 250-kDa FV isoform (FV-short), which can be fully activated by thrombin, is present in all A2440G carriers' plasma (n = 16). FV-short inhibits coagulation through an indirect mechanism by forming a complex with tissue factor pathway inhibitor-α (TFPIα), resulting in an approximately 10-fold increase in plasma TFPIα, suggesting that the TFPIα:FV-short complexes are retained in circulation. The TFPIα:FV-short complexes efficiently inhibit thrombin generation of both intrinsic and extrinsic coagulation pathways. These data demonstrate that the east Texas bleeding disorder-associated F5A2440G leads to the formation of the TFPIα:FV-short complex, which inhibits activation and propagation of coagulation.

Concepts: Blood, Coagulation, Platelet, Protein C, Thrombin, Factor X, Coagulation system, Factor VII

5

Direct inhibitors of coagulation factor Xa (FXa) or thrombin are promising oral anticoagulants that are becoming widely adopted. The ability to reverse their anticoagulant effects is important when serious bleeding occurs or urgent medical procedures are needed. Here, using experimental mouse models of hemostasis, we show that a variant coagulation factor, FXa(I16L), rapidly restores hemostasis in the presence of the anticoagulant effects of these inhibitors. The ability of FXa(I16L) to reverse the anticoagulant effects of FXa inhibitor depends, at least in part, on the ability of the active site inhibitor to hinder antithrombin-dependent FXa inactivation, paradoxically allowing uninhibited FXa to persist in plasma. Because of its inherent catalytic activity, FXa(I16L) is more potent (by >50-fold) in the hemostasis models tested than a noncatalytic antidote that is currently in clinical development. FXa(I16L) also reduces the anticoagulant-associated bleeding in vivo that is induced by the thrombin inhibitor dabigatran. FXa(I16L) may be able to fill an important unmet clinical need for a rapid, pro-hemostatic agent to reverse the effects of several new anticoagulants.

Concepts: Coagulation, Warfarin, Heparin, Factor V, Thrombin, Vitamin K, Factor X, Factor VII