Background: Factor (F)Xa direct oral anticoagulants (DOACs) require rapid reversal in patients with serious bleeding or needing urgent surgery/interventions. VMX-C001 is a recombinant human FX variant under development as an FXa DOAC bypassing agent. Objectives: This study aimed to develop and evaluate the sensitivity of 2 modified commercially available coagulation assays—dilute prothrombin time (dPT) and dilute Russell viper venom time (dRVVT)—to assess coagulation with FXa DOACs with/without VMX-C001. Methods: Citrated platelet-poor plasma samples were obtained from healthy volunteers. Assay evaluation was performed using undiluted and 2.5× diluted plasma for dPT and 3× and 4× diluted plasma for dRVVT in the absence and presence of apixaban (100 or 250 ng/mL) and/or VMX-C001 (30 μg/mL). Additionally, dPT/dRVVT were measured in plasma spiked in vitro with apixaban, edoxaban, or rivaroxaban (0-1600 ng/mL) in the absence and presence of VMX-C001 (15-60 μg/mL), andexanet (50-200 μg/mL), or 4-factor prothrombin complex concentrates (0.25-0.50 IU/mL). In vivo assay evaluation used plasma samples from healthy subjects administered apixaban or rivaroxaban and from nursing home residents receiving FXa DOAC treatment. Results: Both modified dPT and dRVVT assays were highly sensitive to FXa DOACs, showing dose-dependent prolongation of clotting time, which was fully restored when VMX-C001 or andexanet (higher concentrations) was present in the plasma. Using both assays, clotting time from healthy subjects and nursing home residents receiving FXa DOACs correlated strongly with plasma drug concentrations. Conclusion: dPT and dRVVT assays can serve as surrogate end points in studies of VMX-C001 and can also be used to monitor FXa DOAC anticoagulation.

Accurately predicting how point mutations influence protease activity is a major challenge in protein engineering and in the study of coagulation disorders. Factor Xa (FXa) catalyzes the proteolytic activation of prothrombin, a critical step in thrombin generation, yet the molecular details of direct enzyme-substrate recognition and the impact of pathogenic variants remain incompletely defined. Here, we combined AlphaFold-Multimer modeling with explicit-solvent molecular dynamics (MD) simulations to investigate simplified FXa-substrate interactions using prothrombin-derived peptides encompassing the Arg271 and Arg320 cleavage sites in the absence of the full prothrombinase complex. AlphaFold predictions identified catalytically competent peptide orientations, while subsequent MD simulations and free energy analyses identified key contributions of the P1 and P4 cleavage site residues to FXa binding. Distinct stability and affinity differences between the two cleavage sites were highlighted. Active site variants associated with factor X deficiency demonstrated variant-specific disruptions in peptide engagement, implicating key residues within the FXa active site as critical determinants of catalytic activity. Experimental validation with recombinant FXa variants was consistent with the computational predictions, indicating comparable inhibition by the prothrombin peptides. Together, these findings establish an integrative computational-experimental framework for probing local protease-substrate recognition, rationalizing the effects of disease-causing mutations, and guiding the design of therapeutic protease variants.

Direct anticoagulants inhibit coagulation serine proteases by reversibly engaging their active site with high affinity. By modifying the S4 active site subpocket of factor (F)Xa, we previously introduced inhibitor-resistance while preserving catalytic activity. Given the homology between FXa and thrombin in active site architecture and direct anticoagulant binding, we have targeted the S4 subsite to introduce inhibitor resistance in (pro)thrombin. Recombinant prothrombin variants were generated in which I174 was substituted or sequence R92-N98 was exchanged with that of human kallikrein-3. Specific prothrombin clotting activity of the variants was 6-fold (intrinsic clotting) to 10-fold (extrinsic clotting) reduced relative to wild-type prothrombin. Further analyses revealed that modification of the S4 subsite hampers fibrinogen and thrombomodulin-mediated protein C conversion by thrombin. Consistent with this, the thrombin variants displayed a reduced catalytic efficiency towards the peptidyl substrate used in thrombin generation assessments. The variants displayed a 2-fold reduced sensitivity for dabigatran relative to wild-type prothrombin, while argatroban inhibition was unaffected. Analyses using a purified component system revealed an up to 24-fold and 4-fold reduced IC50 for inhibition of thrombin by dabigatran and argatroban, respectively. MD simulations of both dabigatran-bound and unbound (apo) modified thrombin variants indicated these to comprise a larger inhibitor binding pocket relative to wild-type thrombin and display reduced inhibitor binding. As a net effect, (pro)thrombin variants with S4 subsite modifications supported detectable fibrin formation at therapeutic dabigatran concentrations. Our findings provide proof-of-concept for the engineering of thrombin variants that are resistant to direct thrombin inhibitors by modulating the S4 subsite.

Introduction: VMX-C001 is in clinical development to restore coagulation in patients taking factor Xa direct oral anticoagulants (FXa DOACs), who need urgent surgery or have severe bleeding. It is important to ensure patients who require intra-procedural anticoagulation with unfractionated heparin (UFH) and/or post-procedural thromboprophylaxis with low molecular weight heparin (LMWH) can do so after receiving VMX-C001. This study tested the anticoagulant effects of UFH and LMWH administered shortly after VMX-C001 in healthy subjects in situations simulating (a) patients taking FXa DOACs who require restoration of coagulation with VMX-C001 and then require UFH, e.g., for cardiopulmonary bypass; and (b) patients receiving VMX-C001 who then require LMWH for thromboprophylaxis. Methods: This Phase I, single site, open-label study in healthy subjects (aged ≥18–49 years) investigated the effects of VMX-C001, in combination with the FXa DOAC rivaroxaban and alone, on the anticoagulant effect of UFH (UFH cohort) and LMWH (enoxaparin; LMWH cohort), respectively. The UFH cohort received: on day 1, 5000 IU IV UFH; on days 2 to 5, 20 mg oral rivaroxaban once daily; and on day 5, 4 hours (hrs) after rivaroxaban, 170 mg IV VMX-C001 over 10 sec then, after 20 min, 5000 IU IV UFH. The LMWH cohort received: on day 1, 40 mg SC enoxaparin; and on day 4, 170 mg IV VMX-C001 over 10 sec then, after 20 min, 40 mg SC enoxaparin. Descriptive statistics were used. Pharmacodynamic endpoints: Both cohorts: prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and thrombin generation (TG); UFH cohort: activated clotting time (ACT), thrombin time, dilute prothrombin time (dPT), and dilute Russell's viper venom time (dRVVT); LMWH cohort: anti-FXa activity. TG results shown are endogenous thrombin potential with 20 pmol tissue factor; however, full TG results are available. Other endpoints: Included safety (adverse events; AEs) and immunogenicity. Results: In the UFH cohort (N=7), mean age was 28±8 years, 5 (71.4%) were female and all were non-Hispanic white. Following UFH alone on day 1, changes from baseline at 1 hr post-UFH: PT, aPTT and ACT increased, dPT +16.41 sec and dRVVT -2.85 sec; TG decreased to undetectable levels (-100% vs. baseline). Four hrs after rivaroxaban administration on day 4: PT increased, dPT +152.63 sec and dRVVT +89.83 sec; aPTT and ACT showed only small changes and TG -28.55% vs. baseline. On day 5, rivaroxaban had similar effects to day 4. VMX-C001 was administered post-rivaroxaban and, by 15 mins post VMX-C001, restored dPT (-8.67 sec vs. baseline), dRVVT (-3.11 sec vs. baseline) and TG (-6.74% vs. baseline). UFH given after VMX-C001 reduced TG to undetectable levels at 1 hr (-100% vs baseline), as on day 1. In the LMWH cohort (N=8), mean age was 29±6 years, 6 (75.0%) were female and 6 (75.0%) were non-Hispanic white. On day 1, 4 hrs post-LMWH alone, anti-FXa activity greatly increased and TG decreased markedly (-37.94% vs baseline). On day 4, 4 hrs after VMX-C001 followed by LMWH administration, anti-FXa activity greatly increased and TG decreased markedly (-34.17% vs. baseline) and similarly to day 1. PT and aPTT were slightly increased post-VMX-C001 in both cohorts but generally returned to the normal range by 24 hrs post-UFH/LMWH. Treatment-emergent AEs (TEAEs) were reported in 4 (57.1%) and 8 (100%) subjects in the UFH and LMWH cohorts, respectively, were mostly mild and resolved by end of study. The most common TEAE was LMWH injection site pain. There were no reports of severe AEs, serious AEs or TEAEs leading to study drug interruption, withdrawal or premature study discontinuation. A total of 4 of 15 subjects developed anti-VMX-C001 antibodies (titer ≤1:9); however, no abnormalities in PT or aPTT were observed in these subjects at the last follow-up visit (day 31). Anti-FX antibodies were detected in one subject in the UFH cohort at the first follow-up visit, but this was considered a false positive (titer <1:1, no positive anti-VMX-C001 antibody results and no effect on dPT or dRVVT).Conclusion(s): VMX-C001 with or without a FXa DOAC did not impact the anticoagulant activity of UFH or LMWH. This shows VMX-C001 can restore coagulation in people taking FXa DOACs and UFH can be administered immediately after VMX-C001 for anticoagulation during vascular surgery or for cardiopulmonary bypass and LMWH can be used as thromboprophylaxis or anticoagulation in individuals administered VMX-C001.

Background: VMX-C001 is an engineered recombinant human coagulation factor X (FX) insensitive to inhibition by FXa direct oral anticoagulants (FXa-DOACs) which bypasses the effect of FXa DOACs and rapidly restores coagulation upon administration. It is under development for reversal of FXa DOACs in patients with serious bleeding or requiring urgent surgery. Because it acts as a bypassing agent the same dose is expected to be effective regardless of which FXa DOAC a patient has taken. VMX-C001 has previously been investigated in a First in Human study in subjects treated with apixaban and rivaroxaban. This study was performed to extend the range of doses tested with rivaroxaban, to test it in the presence of edoxaban and to test the tolerability of rapid administration. Aims: To assess the safety, tolerability, PK and PD of VMX-C001 in healthy subjects in the absence and presence of FXa DOACs, rivaroxaban and edoxaban, and to test the safety and tolerability of the administration of VMX-C001 over 2 minutes and over 10 seconds. Methods: This is a phase 1, single-center, double-blind, randomized, placebo-controlled study (NCT06372483). In Part 1 a single intravenous dose of VMX-C001 (170 mg; n=6) or placebo (n=2) was administered to a cohort of 8 male or female healthy subjects (age 18-49 years) over 2 minutes. In Part 2, 4 cohorts of 8 healthy older male and female subjects (age 50‒79 years) who had received a 3.5-day course of treatment with rivaroxaban (20 mg once-daily; one cohort) or edoxaban (60 mg once daily, 3 cohorts) were given, 5 min after their last FXa-DOAC dose, a single intravenous dose of VMX-C001 (doses of 113mg to 170mg) or placebo administered over 2 min (3 cohorts, 1 rivaroxaban, 2 edoxaban) or 10 seconds (1 cohort, edoxaban). The local ethics committee approved the study; all volunteers provided written informed consent. Blood samples were collected before and after study drug administration for determination of PK parameters, thrombin generation, diluted prothrombin time (dPT), diluted Russell viper venom time (dRVVT), PT, aPTT and ACT. Safety assessments included adverse events, immunogenicity (antibodies against VMX-C001 or FX), and the levels of D-dimer, and prothrombin F1.2. Results: Subject dosing is completed but the data are still blinded. Overall, 30 subjects received VMX-C001 either alone (n=6) or in combination with FXa-DOACs (n=24). There were no safety or tolerability concerns. Blinded PK data was consistent with previous results and showed dose-proportional linear kinetics with a half-life (t½) of approximately 30 h and a volume of distribution of approximately 9 L. Administration over both 2 minutes and over 10 seconds was well tolerated with no infusion reactions or clinically significant changes in pulse or blood pressure regardless of administration time. Preliminary PD data suggest that VMX-C001 bypasses the anticoagulant effects of rivaroxaban and edoxaban. The complete results will be presented. Conclusion: Previous data shows that VMX-C001 rapidly bypasses the effects of apixaban and rivaroxaban. Preliminary data from this study extends the data on doses of VMX-C001 bypassing rivaroxaban and provides data showing that VMX-C001 bypasses the effect of edoxaban. In this study VMX-C001 was well tolerated, can be administered via a 10 seconds iv push and has a half life of 30 hrs. These data indicate a suitable profile for emergency use and support further clinical development of VMX-C001 for the rapid restoration of coagulation in patients who have taken FXa-DOACs and are experiencing severe bleeding or require urgent surgery.

Background: VMX-C001 is an engineered recombinant human coagulation factor X (FX) that is insensitive to inhibition by FXa direct oral anticoagulants (FXa-DOACs). Upon administration it bypasses the effect of FXa DOACs and rapidly restores coagulation. It is under development for reversal of FXa DOACs in patients with serious bleeding or requiring urgent surgery. VMX-C001 has previously been shown to be safe and well tolerated in a FIH study in subjects treated with apixaban and rivaroxaban. To date, efficacy of VMX-C001 was shown in coagulation assays, such as thrombin generation (TG), dilute prothrombin time (dPT) and dilute Russels' Viper Venom time (dRVVT). An injury-bleeding model was developed in cynomolgus monkeys to study the effects of VMX-C001 on FXa-DOAC induced bleeding in vivo. Aims: To study the effects of VMX-C001, either alone or in combination with a FXa-DOAC (rivaroxaban), on post-injury bleeding in monkeys, and determine how coagulation assays correlate with bleeding. Methods: Male monkeys were anesthetized, the liver was exposed, and a 4 mm diameter/2 mm depth injury was made in the liver using a punch biopsy tool. Post-injury bleeding time was measured up to a maximum of 1200 seconds and blood loss was determined by weighing gauze used to absorb blood from the injury. All animals were treated similarly at baseline. To assess the efficacy of VMX-C001 to bypass the effects of rivaroxaban, post-injury bleeding outcomes were measured in monkeys (N = 24) before and after rivaroxaban administration (IV bolus followed by continuous infusion) alone, and then after VMX-C001 IV bolus administration (1.3 mg/kg; N =7 or 0.9 mg/kg; N =7) or placebo (N = 10). The surgery team and assessors were blinded to the treatment (VMX-C001 or placebo). Post-injury bleeding time and blood loss were assessed at baseline and 20 minutes after each dose. To study the effect of VMX-C001 alone on bleeding outcome monkeys (N = 10) received two IV bolus doses of VMX-C001 (1.3 mg/kg; N = 6 or 3.0 mg/kg; N = 4), 20 minutes apart and bleeding was assessed before and after the first dose, and then after the second dose. Blood samples were taken to evaluate clotting (PT, aPTT, dPT, dRVVT and TG) and VMX-C001 and/or rivaroxaban plasma level. Results: In animals pre-treated with rivaroxaban, clotting assays were prolonged: PT from 9.2±0.6 to 21.7±8.8 sec; aPTT from 23.8±2.4 to 42.0±12.6 sec. VMX-C001 administration shortened the PT in both dose groups (1.3 mg/kg: 14.2±1.2 and 0.9 mg/kg: 16.4±3.3 sec) but the aPTT remained prolonged (1.3 mg/kg: 52.0±6.6 and 0.9 mg/kg: 56.6±6.7 sec). At the time of VMX-C001 administration mean rivaroxaban plasma levels were 554 ng/ml (range 300 - 1732) in the 1.3 mg/kg dose group and 760 ng/ml (range 222 - 1961) in the 0.9 mg/kg group. Mean VMX-C001 plasma levels were 28.4 and 19.4 µg/ml in the 1.3 and 0.9 mg/kg dose groups post administration. At baseline (N = 34), median bleeding time was 144 sec (range 80 - 814) and median blood loss was 2.0 gr (range 0.3 - 9.4). Rivaroxaban increased bleeding time to the maximum of 1200 seconds in 19 out of 24 animals and median blood loss was 14.1 gr (range 2.0 - 56.6). Administration of VMX-C001 reversed the effect of rivaroxaban on bleeding time in both dose groups (1.3 mg/kg: 201 sec, range 123 - 926; 0.9 mg/kg: 239 sec, range 167 - 1133). Additionally, post-injury blood loss was reduced to baseline values in both groups (1.3 mg/kg: 2.3 gr, range 0.5 - 5.3 and 0.9 mg/kg: 2.4 gr, range 1.3 - 8.9). In contrast, bleeding time and blood loss (1200 sec and 21.4 gr, range 10.1 - 76.1) remained high in the placebo group. In monkeys administered VMX-C001 alone, PT marginally increased to 10.5±0.4 sec, but aPTT increased dose-dependently to 36.4±1.4 and 43.8±7.8 sec after two doses of either 1.3 mg/kg or 3.0 mg/kg. After the 2nd dose, mean VMX-C001 plasma levels were 51.0 and 97.4 µg/ml in either group. The increases in PT and aPTT were not associated with altered bleeding outcomes post-injury after dosing twice with 1.3 mg/kg (151 sec, range 91 - 755; 2.1 gr, range 0.2 - 5.8) or 3.0 mg/kg (87 sec, range 69 - 205; 1.7 gr, range 0.8 - 2.7). Conclusion(s): VMX-C001 completely reverses rivaroxaban-induced increases in bleeding time and blood loss, but leaves the aPTT prolonged. VMX-C001 alone has no impact on bleeding outcomes, despite prolonging the aPTT. Changes in PT/aPTT appear not to predict the effect of VMX-C001 on bleeding. Results on coagulation assays TG, dRVVT and dPT are pending and will be communicated at the congress.

Background: 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. Objectives: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. Methods: 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. Results: 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. Conclusion: 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.

Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease ( Gla -deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of h GLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease.

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338L-FIX activity in patients after gene therapy.

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 wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma.

Fabry disease is an X-linked lysosomal storage disorder (LSD) resulting from mutations in the gene encoding for α-galactosidase A (GLA). It is characterised by the abnormal accumulation of neutral glycosphingolipids (GSL), predominantly Globotriaosylceramide (Gb3), in the lysosomes of multiple cell types including vascular endothelial cells. It is associated with early-onset stroke, cardiomyopathy, and progression to end-stage renal failure. We examined the therapeutic efficacy of GLA gene therapy in Fabry mice using recombinant adeno-associated virus (rAAV) vectors, directed by the liver-specific promoter (FRE1). In this study, we showed that a single intravenous (IV) injection of rAAV8-FRE1-GLAco (2x1012 vg/kg) achieved supraphysiological levels of plasma GLA activity up to 1061-fold of normal with concomitant correction of lysosomal storage pathology in multiple key organs in a Fabry mouse model. Animals injected with our novel codon-optimised (“GLAco”) GLA construct exhibited a rapid elevation in plasma GLA activity levels reaching a peak level by 4 weeks post-injection, and this level of expression was maintained for the duration of the study (14 weeks). Furthermore, mass spectrometry (LC-MC/MS) analysis of GSL extract from different tissues provided proof of exposure and storage clearance in various key organs, which is indicative of metabolic cross-correction of liver-derived GLA enzyme. Gb3 levels in the plasma were reduced by 91% (p=0.008) in the kidney by 64% (p=0.045) in the heart by 98% (p<0.0001) in the spleen by 97% (p<0.0001) and in the liver by 99% (p=0.0075) relative to age matched untreated controls. Following a similar trend, Lyso-Gb3 in the plasma were also reduced by 98% (p<0.0001) normalising to that of wild type levels. Gb3 clearance was also supported by electron microscopy data. Collectively, these data provide strong evidence that our liver-directed AAV-mediated gene therapy approach holds considerable therapeutic potential for the treatment of Fabry disease.

Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex.

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.

The direct thrombin and factor Xa (FXa) inhibiting oral anticoagulants have emerged as the preferred anticoagulant therapy, having major benefits for the patient's quality of life due to similar or lower risks of bleeding compared to the vitamin K antagonists. Unfortunately, in the case of bleeding-related adverse effects, no reversal agent is available to counteract the FXa-targeted anticoagulant therapy and restore hemostasis.

Background Obesity is associated with a hypercoagulable state and increased risk for thrombotic cardiovascular events. Objective Establish the onset and reversibility of the hypercoagulable state during the development and regression of nutritionally-induced obesity in mice, and its relation to transcriptional changes and clearance rates of coagulation factors as well as its relation to changes in metabolic and inflammatory parameters. Methods Male C57BL/6J mice were fed a low fat (10% kcal as fat; LFD) or high fat diet (45% kcal as fat; HFD) for 2, 4, 8 or 16 weeks. To study the effects of weight loss, mice were fed the HFD for 16 weeks and switched to the LFD for 1, 2 or 4 weeks. For each time point analyses of plasma and hepatic mRNA levels of coagulation factors were performed after overnight fasting, as well as measurements of circulating metabolic and inflammatory parameters. Furthermore, in vivo clearance rates of human factor (F) VII, FVIII and FIX proteins were determined after 2 weeks of HFD-feeding. Results HFD feeding gradually increased the body and liver weight, which was accompanied by a significant increase in plasma glucose levels from 8 weeks onwards, while insulin levels were affected after 16 weeks. Besides a transient rise in cytokine levels at 2 weeks after starting the HFD, no significant effect on inflammation markers was present. Increased plasma levels of fibrinogen, FII, FVII, FVIII, FIX, FXI and FXII were observed in mice on a HFD for 2 weeks, which in general persisted throughout the 16 weeks of HFD-feeding. Interestingly, with the exception of FXI the effects on plasma coagulation levels were not paralleled by changes in relative transcript levels in the liver, nor by decreased clearance rates. Switching from HFD to LFD reversed the HFD-induced procoagulant shift in plasma, again not coinciding with transcriptional modulation. Conclusions Changes in dietary fat content rapidly alter the mouse plasma coagulation profile, thereby preceding plasma metabolic changes, which cannot be explained by changes in relative expression of coagulation factors or decreased clearance rates.

The prothrombinase complex, which converts prothrombin to the key regulatory enzyme thrombin, consists of the serine protease Xa (FXa) and cofactor Va (FVa; A1-A2-A3-C1-C2 domains). The latter dramatically enhances the rate of prothrombin catalysis and exclusively associates with FXa on negatively charged phospholipid surfaces through its lipid-binding C-domains. Interestingly, we have previously shown that the FVa and FXa homologues found in the venom of the Australian snake Pseudonaja textilis bypass the requirement for a membrane surface to achieve complexation. Here we investigated whether the C-domains of venom-derived P. textilis factor V (ptFV) drive its lipid-independent cofactor function and, if so, can sustain this unique property in the setting of human FV. Therefore, we swapped the C-domains of constitutively active B-domainless human FV (hFV) and ptFV and expressed and purified the chimeric variants hFV-ptC and ptFV-hC. Using a purified prothrombinase assay with the lipid-independent substrate prethrombin-1, FV cofactor activity was examined with the corresponding human or venom-derived P. textilis FXa species in the presence or absence of lipids. We hypothesized that the C-domains constrain ptFV in a conformation optimal for lipid-independent cofactor function. Surprisingly, ptFV-hC displayed full cofactor activity, irrespective of the availability of anionic membranes. Similarly, hFV-ptC functioned equivalent to hFV as no gain-of-function in the absence of lipids was observed. Subsequent lipid-titration experiments indicated that whereas both C-domain species promoted maximum rates of substrate conversion at identical phospholipid concentrations, the lipid-affinity of the ptFV C-domains was at least 10-fold reduced as compared to that of hFV. These results demonstrate that the P. textilis C-domains do not play a role in the unique capacity of ptFV to function in the absence of lipids, suggesting that they may be of little relevance to ptFV cofactor function. As such, our findings imply an alternative mode of macromolecular complex assembly for P. textilis venom FVa and FXa and their natural substrate prothrombin, thereby potentially challenging the current paradigm of human prothrombinase assembly and function.

The venom of the Australian brown snake Pseudonaja textilis contains a prothrombinase-like initiator of blood coagulation, which has evolved into a potent weapon through several gain-of-function adaptations. Here we examined the functional implications of a disulfide bond exclusively found in the factor (F)Va-like cofactor component, ptFV. We found that this remarkable structural feature is not required for the procoagulant properties of ptFV. The nearly identical liver-derived plasma ptFV that lacks this disulfide link displayed a similar procoagulant profile. Whether the unique disulfide bond imposes conformational constraints essential to other aspects of the venom FV life cycle remains to be determined.