The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood ...coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients.
To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models.
We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-), and ...Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation.
Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b(-/-)and Abcb1a/1b;Abcg2(-/-), but not Abcg2(-/-)mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a(-/-), but not Abcb1a/1b(-/-), Abcg2(-/-), or Abcb1a/1b;Abcg2(-/-)mice, displayed highly (>5-fold) increased oral availability of everolimus.
Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies.
Mice deficient in the anticoagulants antithrombin (Serpinc1) or protein C (Proc) display premature death due to thrombosis-related coagulopathy, thereby precluding their use in gene function studies ...and thrombosis models. We used RNA interference to silence Serpinc1 and/or Proc in normal adult mice. The severe coagulopathy that followed combined “knockdown” of these genes is reported. Two days after siRNA injection, thrombi (occlusive) were observed in vessels (large and medium-sized) in multiple tissues, and hemorrhages were prominent in the ocular, mandibular, and maxillary areas. Tissue fibrin deposition and reduction of plasma fibrinogen accompanied this phenotype. The coagulopathy was prevented by dabigatran etexilate treatment. Silencing of Serpinc1 alone yielded a comparable but milder phenotype with later onset. The phenotype was absent when Proc was targeted alone. We conclude that RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes in vivo and provides a novel, controlled mouse model for spontaneous venous thrombosis.
•RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes, alone or in combination, in normal adult mice.•RNA interference of Serpinc1 and Proc provides a novel, controlled mouse model for spontaneous venous thrombosis.
Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α ...and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo.
Single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that harbors the coagulation factor XI (F11), prekallikrein (KLKB1), and a cytochrome P450 family member (CYP4V2) genes are associated with ...deep venous thrombosis (DVT). These SNPs exert their effect on DVT by modifying the circulating levels of FXI. However, SNPs associated with DVT were not necessarily all in F11, but also in KLKB1 and CYP4V2. Here, we searched for evidence for common regulatory elements within the 4q35.2 locus, outside the F11 gene, that might control FXI plasma levels and/or DVT risk. To this end, we investigated the regulation of the orthologous mouse gene cluster under several metabolic conditions that impact mouse hepatic F11 transcription. In livers of mice in which HNF4α, a key transcription factor controlling F11, was ablated, or reduced by siRNA, a strong decrease in hepatic F11 transcript levels was observed that correlated with Cyp4v3 (mouse orthologue of CYP4V2), but not by Klkb1 levels. Estrogens induced hepatic F11 and Cyp4v3, but not Klkb1 transcript levels, whereas thyroid hormone strongly induced hepatic F11 transcript levels, and reduced Cyp4v3, leaving Klkb1 levels unaffected. Mice fed a high-fat diet also had elevated F11 transcription, markedly paralleled by an induction of Klkb1 and Cyp4v3 expression. We conclude that within the mouse F11, Klkb1, Cyp4v3 gene cluster, F11 and Cyp4v3 frequently display striking parallel transcriptional responses suggesting the presence of shared regulatory elements.
Chymotrypsin-like serine proteases are hallmarked by a protease domain comprising the catalytic triad residues His57, Asp102, and Ser195 (chymotrypsinogen numbering) situated in the active site ...cleft. While the catalytic triad in conjunction with the oxyanion hole residues regulate substrate cleavage, the active site subpockets (S1-4) control substrate recognition and binding. The high structural homology of the serine protease domains allows for analogous strategies in drug design, which is underscored by the direct oral anticoagulants (DOACs) for the prophylactic management of stroke in atrial fibrillation and prevention and treatment of venous thrombosis. DOACs inhibit coagulation serine proteases by reversibly engaging the active site with high affinity. To expand the repertoire of DOAC-specific reversal agents we have previously successfully modified the S4 active site subpocket of human factor Xa to prevent DOAC binding while preserving catalytic activity Verhoef 2017 Nature Commun.. To explore whether an analogous strategy can be applied to create DOAC resistance in the serine protease thrombin, specific substitutions or sequences in or around the dabigatran-binding S4 subsite derived from naturally occurring serine proteases or plasma proteins were introduced in prothrombin.
A panel of prothrombin variants was generated and transfected into HEK293 cells to allow for stable protein expression. In some of the generated prothrombin variants comprising insertions in amino acid sequence 91-99 that is directly adjacent to the S4 subsite protein expression was severely impaired. This indicates that exchange of any surface-exposed serine protease or plasma protein region into the prothrombin protease domain is not necessarily compatible with protein expression. In contrast, exchange of the human prothrombin 91-99 sequence for that of human kallikrein 3 or targeted amino acid replacement of S4 subsite residue Ile174 resulted in prothrombin protein expression levels similar to wild-type prothrombin.
Following expression, prothrombin variants were purified to homogeneity using the CaptureSelect tm affinity matrix that selects for fully gamma-carboxylated prothrombin. The specific prothrombin clotting activity analyses of the purified prothrombin variants KL3 (0.7 ± 0.2 U/mg), I174A (0.8 ± 0.2 U/mg), and I174F (0.8 ± 0.3 U/mg) demonstrated an overall ~10-fold reduced specific activity relative to wild-type prothrombin (7.5 ± 0.1 U/mg). As such, modification of the S4 subsite likely interferes with the binding and subsequent conversion of fibrinogen by thrombin. To determine whether the prothrombin variants supported tissue factor-initiated thrombin formation in human plasma, prothrombin-deficient plasma was supplemented with increasing plasma concentrations of prothrombin variant (90-180 ug/mL). Consistent with their reduced specific clotting activity, 180 ug/mL prothrombin variant was required to obtain substantial thrombin generation but with reduced thrombin generation parameters (peak thrombin, ETP) relative to supplementation with plasma concentrations of wild-type prothrombin (90 ug/mL). This calibrated automated thrombin generation assay set-up was used to assess the DOAC-resistance of the prothrombin variants. While thrombin formation reached half-maximum inhibition at 532 ± 58 nM dabigatran in wild-type prothrombin-supplemented plasma, addition of the prothrombin variants displayed a ~2-fold reduced sensitivity to dabigatran inhibition (IC50: 1186 ± 136 nM prothrombin-KL3; 851 ± 97 nM prothrombin-I174F; 772 ± 80 nM prothrombin-I174A). This demonstrates that the S4 subsite-modified prothrombin variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor.
Collectively, our findings indicate that human prothrombin variants comprising a single point mutation at position Ile174 in the S4 subsite or at a region directly adjacent to the S4 subsite are able to generate thrombin in plasma inhibited by dabigatran. Hence, serine proteases with S4 subpocket modifications have the potential to bypass DOAC therapy and could provide a generic strategy in the development of novel DOAC-bypassing agents.
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Reitsma: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Verhoef: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.
Factor (F)IX-FIAV, a FIX variant with four amino acid substitutions that functions independently of the cofactor VIIIa, has been previously shown to ameliorate the hemophilia A (HA) phenotype in vivo ...Quade-Lyssy et al. J. Thromb. Haemost. 2014. Here we evaluated the efficacy of purified recombinant FIX-FIAV in severe, moderate, and mild hemophilia A patient plasma employing thrombin generation and intrinsic clotting activity (aPTT) analyses. The combination of FIX-FIAV with current hemophilia A therapeutics was used for preclinical safety assessment.
Plasma was obtained from 21 HA patients, seven per HA phenotype, with a median age of 38 years interquartile range (IQR) 27.5 - 49.5. The plasma levels of FIX, FX, prothrombin, and antithrombin of all included patients were within the normal range. To determine the effect of FIX-FIAV on FXIa-triggered thrombin generation parameters (lag time, endogenous thrombin potential (ETP)), plasma was spiked with 100% (5 µg/ml in severe/mild) or 125% (6 µg/ml in moderate) FIX-FIAV prior to analysis. FIX-FIAV significantly shortened the lag time in all patient plasmas irrespective of disease severity (Figure 1) with an overall median of 4.4 min IQR 3.6 - 6.9 in the absence of FIX-FIAV vs. 3.1 min IQR 2.4 - 4.5 in the presence of FIX-FIAV (p<0.0001, Table 1). Similar observations were obtained following aPTT analyses: median clotting time of 115.5 sec IQR 105 - 173.9 without vs. 97.7 sec IQR 91.8 - 136.2 with 100% FIX-FIAV (p = 0.0039). Conversion of the thrombin generation lag time to FVIII-like activity using a FVIII calibration for each individual patient plasma revealed that FIX-FIAV mitigated the HA phenotype from severe to moderate, from moderate to mild, and from mild to normal (Table 1).
Interestingly, following the addition of FIX-FIAV a minor but significant decrease in ETP was observed for non-severe HA patient plasma (p = 0.016 for mild and p = 0.016 for moderate), while FIX-FIAV increased the median ETP in severe HA plasma by 2.1-fold (p = 0.22) (Figure 2, Table 1). This may result from competition between the added FIX-FIAV and endogenous FIX for interaction with residual functional FVIII. In line with this, experiments performed in the presence of an anti-FVIII antibody that inhibits FVIII activity significantly enhanced the ETP in all patient plasmas (p = 0.016 for each individual severity, Figure 2) in addition to shortening the lag time (Figure 1).
Next, we evaluated the combination of FIX-FIAV with bypassing agents (1 IU/mL aPCC or 1.5 mg/mL rFVIIa) in nine patient plasmas, three per phenotype. Addition of aPCC or rFVIIa to FIX-FIAV-spiked plasma did not significantly affect aPTT clotting times nor ETP values in comparison to adding aPCC or rFVIIa only, respectively. The median lag time shortened significantly, albeit modestly, for the combination of FIX-FIAV with aPCC in comparison to conditions with aPCC only: 5.2 min IQR 3.0 - 6.4 vs. 5.5 min IQR 3.6 - 7.7, p = 0.0078, respectively. Similar findings were obtained when combining FIX-FIAV with rFVIIa relative to rFVIIa only: median lag time 4.9 min IQR 2.2 - 6.1 vs. 5.3 min IQR 3.3 - 8.8, p = 0.0039. Hence, no substantial synergistic effect was observed when combining FIX-FIAV with bypassing agents aPCC or rFVIIa for HA. In contrast, combining approximate hemostatic levels (55 µg/ml) of emicizumab, a bispecific antibody mimicking FVIIIa, with FIX-FIAV resulted in a ~1.1-fold reduced lag time and ETP relative to emicizumab alone (p = 0.016 and p = 0.004 respectively). This is suggestive of a minor synergistic procoagulant effect, which is consistent with the FIX(a)-FX(a) bridging capacity of emicizumab.
In conclusion, FIX-FIAV could serve as a potential treatment for hemophilia A as it mitigates the hemophilia A phenotype in patient plasma, also in the presence of an inhibitory anti-FVIII antibody. While further safety assessment is warranted, no severe procoagulant effects were observed for the combination of FIX-FIAV with conventional hemophilia A therapeutics.
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Romano: Swedish Orphan Biovitrum B.V.: Other: Travel grant and aforementioned Research Funding in the form of the Young Investigator's Award 2020, Research Funding. Liu: uniQure Biopharma B.V.: Current Employment. McCreary: uniQure Biopharma B.V.: Ended employment in the past 24 months. Leebeek: Roche: Other: DSMB member of a study sponsored by Roche; uniQure Biopharma B.V.: Consultancy, Research Funding; Swedish Orphan Biovitrum B.V.: Other: Travel support, Research Funding; Biomarin: Consultancy; CSL Behring: Consultancy, Research Funding; Shire/Takeda: Consultancy, Research Funding. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.
The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical ...protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV Quade-Lyssy et al. J. Thromb. Haemost. 2014. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo.
To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV.
Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (<13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven.
Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A.
Strijbis:uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, ...bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity.
The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed.
Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography.
F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients.
These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.
Recombinant factor (F)IX-FIAV has previously been shown to function independently of activated FVIII (FVIIIa) and ameliorate the hemophilia A (HA) phenotype in vitro and in vivo.
The aim of this ...study was to assess the efficacy of FIX-FIAV in plasma from HA patients using thrombin generation (TG) and intrinsic clotting activity (activated partial thromboplastin time APTT) analyses.
Plasma obtained from 21 patients with HA (>18 years; 7 mild, 7 moderate, and 7 severe patients) was spiked with FIX-FIAV. The FXIa-triggered TG lag time and APTT were quantified in terms of FVIII-equivalent activity using FVIII calibration for each patient plasma.
The linear, dose-dependent improvement in the TG lag time and APTT reached its maximum with approximately 400% to 600% FIX-FIAV in severe HA plasma and with approximately 200% to 250% FIX-FIAV in nonsevere HA plasma. The cofactor-independent contribution of FIX-FIAV was therefore suggested and confirmed by the addition of inhibitory anti-FVIII antibodies to nonsevere HA plasma, resulting in a FIX-FIAV response similar to severe HA plasma. Addition of 100% (5 μg/mL) FIX-FIAV mitigated the HA phenotype from severe to moderate (from <0.01% to 2.9% IQR 2.3%-3.9% FVIII-equivalent activity), from moderate to mild (3.9% IQR 3.3%-4.9% to 16.1% IQR 13.7%-18.1% FVIII-equivalent activity), and from mild to normal (19.8% IQR 9.2%-24.0% to 48.0% IQR 34.0%-67.5% FVIII-equivalent activity). No substantial effects were observed when combining FIX-FIAV with current HA therapies.
FIX-FIAV is capable of increasing the FVIII-equivalent activity and coagulation activity in plasma from HA patients, thereby mitigating the HA phenotype. Hence, FIX-FIAV could serve as a potential treatment for HA patients with or without inhibitors.
•In previous studies, factor (F) IX-FIAV improved the hemophilia A phenotype in vitro and in vivo.•FVIII-equivalent activity of FIX-FIAV was tested in (non-)severe hemophilia A plasma.•FIX-FIAV improves thrombin formation and mitigates the hemophilia A phenotype in plasma.•FIX-FIAV could serve as a potential hemophilia A treatment regardless of inhibitor status.
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