Human Factor Xa Research Articles

From Venom to Vein: Factor VII Activation as a Major Pathophysiological Target for Procoagulant Australian Elapid Snake Venoms.

Australian elapid snake venoms are uniquely procoagulant, utilizing blood clotting enzyme Factor Xa (FXa) as a toxin, which evolved as a basal trait in this clade. The subsequent recruitment of Factor Va (FVa) as a toxin occurred in the last common ancestor of taipans (Oxyuranus species) and brown snakes (Pseudonaja species). Factor II (prothrombin) activation has been stated as the primary mechanism for the lethal coagulopathy, but this hypothesis has never been tested. The additional activation of Factor VII (FVII) by Oxyuranus/Pseudonaja venoms has historically been considered as a minor, unimportant novelty. This study aimed to investigate the significance of toxic FVII activation relative to prothrombin activation by testing a wide taxonomical range of Australian elapid species with procoagulant venoms. The activation of FVII or prothrombin, with and without the Factor Va as a cofactor, was assessed, along with the structural changes involved in these processes. All procoagulant species could activate FVII, establishing this as a basal trait. In contrast, only some lineages could activate prothrombin, indicating that this is a derived trait. For species able to activate both zymogens, Factor VII was consistently more strongly activated than prothrombin. FVa was revealed as an essential cofactor for FVII activation, a mechanism previously undocumented. Species lacking FVa in their venom utilized endogenous plasma FVa to exert this activity. The ability of the human FXa:FVa complex to activate FVII was also revealed as a new feedback loop in the endogenous clotting cascade. Toxin sequence analyses identified structural changes essential for the derived trait of prothrombin activation. This study presents a paradigm shift in understanding how elapid venoms activate coagulation factors, highlighting the critical role of FVII activation in the pathophysiological effects upon the coagulation cascade produced by Australian elapid snake venoms. It also documented the novel use of Factor Va as a cofactor for FVII activation for both venom and endogenous forms of FXa. These findings are crucial for developing better antivenoms and treatments for snakebite victims and have broader implications for drug design and the treatment of coagulation disorders. The research also advances the evolutionary biology knowledge of snake venoms.

The use of andexanet alpha in the Polish setting: An interdisciplinary protocol. Expert consensus statement of the Polish Cardiac Society.

Andexanet alfa (AA) is a recombinant inactive analog of human activated factor X (FXa), effectively reversing the effects of its inhibitors - rivaroxaban and apixaban, which are available in Poland. The drug was approved for clinical use registration after the publication of the results of the ANNEXA-4 trial (Andexanet Alfa, a Novel Antidote to the Anticoagulation Effects of FXa Inhibitors 4), in which its efficacy in restoring hemostasis in life-threatening hemorrhages in patients receiving using the aforementioned anticoagulants was demonstrated. Hence, AA is now recommended for patients on apixaban or rivaroxaban therapy with massive and uncontrollable hemorrhages, including hemorrhagic strokes (HS) and gastrointestinal bleeding. Drug-specific chromogenic anti-Xa assays are generally best suited for estimating rivaroxaban and apixaban plasma levels, aside from direct assessment of their concentrations. The absence of anti-Xa activity, determined using these assays, allows us to rule out the presence of clinically relevant plasma concentrations of any FXa inhibitor. On the other hand, the dose of AA should not be modified based on the results of coagulation tests, as it depends solely on the time that elapsed since the last dose of FXa inhibitor and oon the dose and type of FXa inhibitor. AA is administered as an intravenous (i.v.) bolus, followed by an i.v. infusion of the drug. The maximum reversal of anti-Xa activity occurs within two minutes of the end of the bolus treatment, with the continuation of the continuous i.v. infusion allowing the effect to be maintained for up to two hours afterwards. Because anticoagulant activity can reappear after the infusion is completed, it is currently unclear at what point after AA administration FXa inhibitors or heparin should be re-administered. In Poland AA is starting to become available and its urgent need to administer it to patients with severe bleeding on apixaban or rivaroxaban.

Recommendations for the use of andexanet alfa in the management of bleeding in patients on oral factor Xa inhibitors in Switzerland: Guideline from the Working Party Hemostasis of the Swiss Society of Hematology

Anticoagulants are essential in preventing and treating thrombosis. Unfortunately, their use is accompanied by an enhanced risk of bleeding. Since the introduction of direct oral anticoagulants (DOACs), the risk of major bleeding has been reduced but not eliminated. Major bleeding events related to the use of factor Xa inhibitors can be challenging to manage. In recent years, four-factor prothrombin complex concentrates have been used in patients with severe bleeding taking oral direct factor Xa inhibitors (apixaban, edoxaban and rivaroxaban). Andexanet alfa (OndexxyaTM, AstraZeneca AG) is a specially designed recombinant version of human factor Xa that acts as a decoy receptor to reverse the effects of factor Xa inhibitors. Since 2 December 2020, andexanet alfa has been used in Switzerland for adult patients receiving apixaban or rivaroxaban when reversal of anticoagulation is required because of life-threatening or uncontrolled bleeding. However, the use of andexanet alfa remains a challenge owing to its cost, the reported thrombotic complications and the fact that its efficacy mainly relates to intracranial haemorrhage. Moreover, the use of nonspecific reversal agents together with andexanet alfa is controversial. The present recommendations on the use of andexanet alfa in the management of bleeding in patients on factor Xa inhibitors in Switzerland were developed by a group of Swiss experts from the Working Party Hemostasis of the Swiss Society of Hematology. These recommendations aim to provide support to clinicians in their decision-making in the management of patients with major bleeding receiving factor Xa inhibitors.

Guianensin, a Simulium guianense salivary protein, has broad anti-hemostatic and anti-inflammatory properties.

Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation. Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice. The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.

Andexanet alfa (ONDEXXYA<sup>®</sup> for Intravenous Injection 200 mg), a reversal agent for direct factor Xa inhibitors: pharmacological characteristics and clinical evidence

Andexanet alfa is a modified recombinant human factor Xa (FXa) that was designed to serve as a binding target for FXa inhibitors as decoy protein. It sequesters FXa inhibitors from binding to endogenous FXa, thereby reversing anticoagulant effect of FXa inhibitors. Andexanet alfa has been approved in March 2022 in Japan for patients with life-threatening or uncontrolled bleeding while on treatment with a FXa inhibitor, apixaban, rivaroxaban, or edoxaban tosilate hydrate. It is administered via two dosing regimens, based on the type of FXa inhibitor, dose, and time since the last dose. In nonclinical studies, andexanet alfa significantly inhibited bleeding induced by FXa inhibitors in animal bleeding models. In the development for Japanese patients, the following two clinical studies have been conducted to confirm the efficacy and safety. First, safety and the reversal effect of andexanet alfa on the FXa inhibitor-mediated anticoagulant activity in healthy adults were confirmed in the overseas phase 2 study including Japanese subjects. Next, the reversal effect of andexanet alfa on the anticoagulation activity and the hemostasis were demonstrated in patients with acute major bleeding while on FXa inhibitor treatment in the global phase 3b/4 study (ANNEXA-4 study). The subgroup analysis of Japanese population showed that the efficacy and safety results were consistent with those of overall population. Andexanet alfa is the first approved reversal agent for FXa inhibitors in Japan and is expected to contribute to the improvement of prognosis in patients with fatal and/or uncontrolled bleeding by timely reversing anticoagulant effect of FXa inhibitors.

Application of Activators Ecarin and Factor Xa in Thrombelastography for Measurement of Anticoagulant Effect of Direct Oral Anticoagulants Using TEG 5000.

There are situations where monitoring direct oral anticoagulants (DOACs) would be useful, including bleedings and trauma. The thromboelastographic technique has proven useful in bleeding situations in trauma and heart surgery. The aim of this study was to examine the effect of DOACs on all currently commercially available conventional TEG®5000 assays as well as novel modified assay using Ecarin and human factor Xa (HFXa). Healthy male volunteers were given single dose of oral dabigatran 150 mg, rivaroxaban 20 mg, or apixaban 5 mg. Kaolin, RapidTEG, functional fibrinogen, PlateletMapping assay, and novel modified assays using Ecarin and HFXa were prepared. All TEG parameters were recorded. DOAC concentrations were correlated to the parameters with highest response to the DOAC effect. Sensitivity and negative predictive value of the parameter with highest response to DOAC concentration of 50 ng/mL was calculated. None of the conventional TEG assays demonstrated significant response to the effect on apixaban. Using Ecarin, reaction time R was strongly correlated with dabigatran concentrations. Using HFXa assay, R was strongly correlated with rivaroxaban and apixaban concentrations: r = 0.96, 0.84, and 0.86, respectively; p < 0.0001 for all. The R times obtained with the modified assays demonstrated strong sensitivity and negative predictive values for DOAC levels of ≥50 ng/mL. We have demonstrated that TEG®5000 can monitor the DOAC effect on hemostasis when the appropriate activator is used with significant correlation with DOAC concentrations. Larger clinical studies are warranted for correlation of TEG profile and clinical outcomes.

Andexanet Alfa for Reversal of Factor Xa Inhibitors in Intracranial Hemorrhage: Observational Cohort Study.

Background: Intracranial hemorrhage (ICH) is associated with high mortality and morbidity, especially in patients under anticoagulative treatment. Andexanet alfa (AA) is a modified recombinant form of human factor Xa (FXa) developed for reversal of FXa-inhibitors, e.g., in the event of ICH, but experience is still limited. Methods: This monocentric retrospective observational cohort study included 46 patients with acute FXa-inhibitor-associated non-traumatic ICH (FXa-I-ICH) of whom 23 were treated with AA within 12 h after symptom onset, compared to 23 patients with usual care (UC). Volumetrically analyzed hematoma expansion (HE) in brain imaging, clinical outcome and incidence of adverse events were analyzed. Results: All patients (mean age 79.8 ± 7.2 years) were effectively anticoagulated. The cohort included severely ill patients with large hematoma volumes (median 20.4, IQR 7.8–39.0 mL). Efficacy, as assessed by HE in imaging, was very good in the AA-group. There was no (0.0%) relevant HE (>33%) in contrast to UC-group (26.1%). Nevertheless, we observed a high incidence of thromboembolic events (30.4% vs. 4.4%) and non-favorable outcomes (death/palliative condition) in 43.5% vs. 26.1%. Conclusions: There was no HE in the volumetric neuroimaging assessment in the AA-group, but clinical outcomes remained often worse. Large randomized trials for the use of AA in patients with acute FXa-inhibitor-associated ICH are needed to investigate the clinical outcome in consideration of the rates of thromboembolism.

Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus, Displays Anti-FXa-PAR Signaling In Vitro and In Vivo.

Blood-feeding arthropods secrete potent salivary molecules, which include platelet aggregation inhibitors, vasodilators, and anticoagulants. Among these molecules, Alboserpin, the major salivary anticoagulant from the mosquito vector Aedes albopictus, is a specific inhibitor of the human coagulation factor Xa (FXa). In this study, we investigated the anti-inflammatory properties of Alboserpin, in vitro and in vivo. In vitro, Alboserpin inhibited FXa-induced protease-activated receptor (PAR)-1, PAR-2, PAR-3, VCAM, ICAM, and NF-κB gene expression in primary dermal microvascular endothelial cells. Alboserpin also prevented FXa-stimulated ERK1/2 gene expression and subsequent inflammatory cytokine release (MCP-1, TNF-α, IL-6, IL-8, IL-1β, IL-18). In vivo, Alboserpin reduced paw edema induced by FXa and subsequent release of inflammatory cytokines (CCL2, MCP-1, IL-1α, IL-6, IL-1β). Alboserpin also reduced FXa-induced endothelial permeability in vitro and in vivo. These findings show that Alboserpin is a potent anti-inflammatory molecule, in vivo and in vitro, and may play a significant role in blood feeding.

In silico evaluation of caffeic acid from coconut (Cocos nucifera L.) husks as a potential inhibitor of the human factor Xa

Selectively inhibiting the FXa has a broad therapeutic window as an anticoagulant target because of its starting position of the common pathway of the coagulation cascade which effectively blocks the coagulation. This study investigated the inhibiting capabilities of caffeic acid present in C. nucifera L. husks on the FXa and explored its ADMET profile using bioinformatic predicting tools. The caffeic acid and FXa structure was retrieved from PubChem database and RCSB Protein Data Bank, respectively. Binding geometries were illustrated with the use of AutoDock MGL Tools, AutoDock Vina, PyMol, and the ADMET profile was predicted with ADMETlab 2.0. Results of the in silico methods showed that caffeic acid interacted with residues within the active center of FXa, blocking the access of its native substrates, and demonstrated acceptable pharmacokinetics and drug-like effects, thus it can be recommended for the drug design and development of FXa inhibitors.

Human Prothrombin Variants with Modifications in the Active Site S4 Subpocket Demonstrate Resistance to Dabigatran

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. [Display omitted] DisclosuresReitsma: 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.

Outcomes of activated prothrombin complex concentrate for direct Xa inhibitor bleeding

Bleeding due to oral anticoagulant therapy is recognized as a high priority drug safety problem [ [1] FDA Adverse Events Reporting System (FAERS)Latest Quarterly Data Files (2017) Food and Drug Administration Web Site. 2021 Google Scholar ]. As the use of direct Xa inhibitors continues to expand, the management of bleeding is an important issue. Andexanet, a modified human factor Xa decoy reversal agent for apixaban or rivaroxaban associated bleeding, is available in some countries however concerns with its use have been expressed [ [2] Peled H. Dau N.Q. Lau H. Key points to consider when evaluating andexxa for formulary addition. Neurocrit. Care. 2020; 33: 20-24 Crossref PubMed Scopus (7) Google Scholar ]. Four factor prothrombin complex concentrate (4FPCC) is an alternative reversal agent which has been used off-label for direct Xa inhibitor bleeding [ 3 Steffel J. et al. The 2018 european heart rhythm association practical guide on the use of non-vitamin K antagonist oral anticoagulants in patients with atrial fibrillation. Eur. Heart J. 2018; 39: 1330-1393 Crossref PubMed Scopus (1060) Google Scholar , 4 Witt D.M. et al. American Society of Hematology 2018 guidelines for management of venous thromboembolism: optimal management of anticoagulation therapy. Blood Adv. 2018; 2: 3257-3291 Crossref PubMed Scopus (188) Google Scholar ]. Four factor PCC is available either as an activated or non-activated product. Limited clinical data exist evaluating activated 4FPCC, FEIBA [anti-inhibitor coagulant complex®, Baxalta US Inc., Lexington MA, USA] as a reversal agent [ 5 Engelbart J.M. et al. Safety and effectiveness of factor eight inhibitor bypassing activity for direct oral anticoagulant-related hemorrhage reversal. Am. J. Emerg. Med. 2019; 37: 214-219 Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar , 6 Mao G. et al. Factor eight inhibitor bypassing agent (FEIBA) for reversal of target-specific oral anticoagulants in life-threatening intracranial bleeding. J. Emerg. Med. 2017; 52: 731-737 Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar ]. As andexanet is not on formulary, the reversal protocol at our institution recommended the use of activated 4FPCC 25–50 IU/kg for direct Xa inhibitor bleeding for situations where reversal is desired. Our goal was to assess the patient characteristics, management and outcomes of activated 4FPCC for reversal of apixaban or rivaroxaban acute bleeding events.

Impact of Idarucizumab and Andexanet Alfa on DOAC Plasma Concentration and ClotPro® Clotting Time: An Ex Vivo Spiking Study in A Cohort of Trauma Patients.

Specific antagonists have been developed for the reversal of direct oral anticoagulants (DOAC). We investigated the impact of these reversal agents on the plasma concentration and visco-elastic test results of dabigatran and factor Xa inhibitors. After baseline measurements of dabigatran, the plasma concentration, and the visco-elastic ClotPro® ecarin clotting time (ECA-CT), we added the reversal agent Idarucizumab in vitro and these two analyses were repeated. Likewise, the baseline plasma concentration of apixaban, edoxaban, and rivaroxaban as well as ClotPro® Russell’s viper venom test clotting time (RVV-CT) were measured and reanalyzed following Andexanet alfa spiking. We analyzed fifty blood samples from 37 patients and 10 healthy volunteers. Idarucizumab decreased the measured dabigatran plasma concentration from 323.9 ± 185.4 ng/mL to 5.9 ± 2.3 ng/mL and ECA-CT from 706.2 ± 344.6 s to 70.6 ± 20.2 s, (all, p < 0.001). Andexanet alfa decreased the apixaban concentration from 165.1 ± 65.5 ng/mL to 9.8 ± 8.1 ng/mL, edoxaban from 152.4 ± 79.0 ng/mL to 36.4 ± 19.2 ng/mL, and rivaroxaban from 153.2 ± 111.8 ng/mL to 18.1 ± 9.1 ng/mL (all p < 0.001). Andexanet alfa shortened the RVV-CT of patients with apixaban from 239.2 ± 71.7 s to 151.1 ± 30.2 s, edoxaban from 288.2 ± 65.0 s to 122.7 ± 37.1 s, and rivaroxaban from 225.9 ± 49.3 s to 103.7 ± 12.1 s (all p < 0.001). In vitro spiking of dabigatran-containing blood with Idarucizumab substantially reduced the plasma concentration and ecarin-test clotting time. Andexanet alfa lowered the concentration of the investigated factor Xa-inhibitors but did not normalize the RVV-CT. In healthy volunteers’ blood, Idarucizumab spiking had no impact on ECA-CT. Andexanet alfa spiking of non-anticoagulated blood prolonged RVV-CT (p = 0.001), potentially as a consequence of a competitive antagonism with human factor Xa.

Phe174-Mutated Human Factor X As Bypassing Agent to the Direct FXa Inhibitors

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.The direct FXa inhibitors effectively block its catalytic activity by reversible high affinity occupation of the FXa active site. X-ray crystal structures of human FXa in complex with the FXa inhibitors apixaban or rivaroxaban revealed their specific binding mode and demonstrated occupation of the FXa substrate binding S1 and S4 subsites. To better understand the molecular requirements for direct inhibitor binding in FXa, we performed 750 ns Molecular Dynamics (MD) simulations of the FXa-apixaban complex. During MD, the narrow distribution of close range contacts (≤ 0.5 nm) between the side chain atoms of the S4 pocket residues Tyr99 and Phe174 (chymotrypsinogen numbering) and apixaban confirmed stabilization of apixaban in the S4 subsite. To directly assess the contribution of S4 pocket residues Tyr99 and Phe174 to apixaban binding, we generated human FX(a) variants comprising either Tyr99Ala, Phe174Ala, or both. While substitution of either one of these residues reduced the sensitivity for apixaban by ~8-fold, replacement of both increased the IC50 >100-fold (5 nM wt-FXa; 34 nM Y99A-FXa; 45 nM F174A-FXa; 616 nM Y99A/F174A-FXa). The FX clotting activity, on the other hand, was drastically reduced by substituting Tyr99 (6% residual activity) and essentially lost with both replacements. In contrast, following Phe174 replacement full FX clotting activity was maintained. Since this points to Phe174 substitution as a promising target for abrogated inhibitor binding while maintaining FX activity, we continued with assessing the efficacy of Phe174-mutated FX variants as bypassing agent for the direct FXa inhibitors.To do so, the human variants FX-F174A and FX-F174S were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. Evaluation of the specific FX clotting activity revealed that the Phe174-substituted FX variants exhibited a similar clotting activity (84 mU/µg FX-F174A; 122 mU/µg FX-F174S) relative to human wild-type (wt-)FX (105 mU/µg). This indicates that substitution of Phe174 does not interfere with the binding and subsequent conversion of the FXa substrate. Inhibition of the RVV-X-activated FX variants employing peptidyl substrate conversion revealed a reduced sensitivity to apixaban, demonstrated by a 10-fold increase in IC50 (2 nM wt-FX; 19 nM FX-F174A; 20 nM FX-F174S). Consequently, a decrease in sensitivity was also observed for inhibition by rivaroxaban (3-8-fold) and edoxaban (4-7-fold). To determine whether the FX variants supported tissue factor-initiated (6 pM) thrombin formation in normal human plasma, we performed FXa inhibitor titrations using the calibrated automated thrombin generation assay. While thrombin formation reached half-maximum inhibition at 55 nM apixaban in wt-FX-spiked plasma, plasma spiked with the FX variants displayed a ~10-fold reduced sensitivity to apixaban inhibition (IC50: 640 nM FX-F174S; 405 nM FX-F174A). A similar loss of inhibitor-sensitivity was observed for inhibition by rivaroxaban (6-10-fold) and edoxaban (17-fold), demonstrating that the Phe174-mutated FX variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor. Furthermore, at supra-physiological inhibitor concentrations (1 µM), little if any thrombin was formed upon adding up to 60 µg/ml wt-FX. In contrast, supplementation with 30-45 µg/ml Phe174-mutated FX restored the thrombin peak up to 80% in edoxaban-treated plasma, while the thrombin peak was fully restored in the presence of apixaban or rivaroxaban.Collectively, our findings indicate that human FX variants comprising a single point mutation at position Phe174 in the S4 subsite are able to restore hemostasis in plasma inhibited by the direct FXa inhibitors. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of the direct FXa inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresVerhoef:Leiden University Medical Center: Patents & Royalties: co-inventor on a patent application related to this abstract. Reitsma:Leiden University Medical Center; VarmX B.V.: Equity Ownership, Patents & Royalties: co-inventor on a patent application related to this abstract. Bos:Leiden University Medical Center: Patents & Royalties: co-inventor on a patent application related to a submitted abstract; Bayer: Other: Fee for Presenting at a Bayer Conference; Bayer Hemophilia Awards: Research Funding.

Evolutionary adaptation of the γ‐carboxyglutamic acid domain in blood coagulation factor X in the Serpentes suborder

The serine protease factor Xa plays an important role in blood coagulation as it, in complex with its cofactor Va, proteolytically activates prothrombin to thrombin, the latter being a key enzyme in coagulation. To perform this vital function, factor Xa undergoes numerous posttranslational modifications including vitamin K-dependent γ-carboxylation of glutamic acid (Glu) residues into so-called Gla residues. A correctly γ-carboxylated GLA domain is essential for interaction with the negatively charged membrane surface of activated platelets or endothelial cells at the site of injury where clot formation is required. Moreover, assembly of factor Xa with the cofactor Va into the prothrombinase enzyme complex occurs on a negatively charged membrane surface only. Interestingly, the venom of some Australian Elapid snakes comprises powerful prothrombin-activating enzyme complexes consisting of factor Xa- and Va-like proteins that are specifically expressed in the venom gland. We have previously demonstrated that this venom enzyme complex is capable of functioning independently of a negatively charged phospholipid membrane. We aim to uncover the putative differential role of the GLA domain between human and snake venom (Pseudonaja textilis, pt) factor Xa. To do so, we exchanged the human factor Xa (hFX) GLA domain for that of the snake venom ortholog (ptFX), thereby generating hFX-wt, hFX-ptGLA, ptFX-wt, ptFX-hGLA. The FX variants were recombinantly expressed and purified employing chromatography techniques. FX activation by either the human extrinsic or intrinsic coagulation tenase complex was analyzed using a peptidyl substrate specific for activated FX. Introduction of the ptGLA domain into hFX modestly improved the catalytic rate of the extrinsic reaction, while for the intrinsic reaction the substrate binding affinity was enhanced. Conversely, using similar conditions no FXa formation could be detected following incubation of ptFX or ptFX-hGLA with the human intrinsic or extrinsic tenase complex. Assessment of the FX-specific plasma clotting activity in which thrombin-dependent fibrin clot formation is monitored following either an extrinsic or intrinsic coagulation trigger revealed that while the extrinsic clotting activity of hFX was drastically reduced when hGLA was exchanged for ptGLA (3% residual activity), its intrinsic activity was unperturbed. Use of human versus rabbit reagents for the extrinsic pathway may explain the different observations. Conversely, exchange of ptGLA for hGLA resulted in an overall substantial increase in the FX-specific clotting activity of ptFX-hGLA. Finally, exchanging the ptGLA domain for the hGLA domain did not abolish the requirement for a negatively charged membrane surface in Va-dependent prothrombin activation. These data indicate that the snake venom GLA domain interacts with the intrinsic tenase complex and contributes to substrate conversion and subsequent thrombin formation, which may point to evolutionary adaptation of this snake venom GLA domain.

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors.

Background:In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation.Methods:In order to search for tools to improve the antivenom’s, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes.Results:Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity.Conclusion:Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic β-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1–4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

The mode of action of sugammadex on coagulation.

To explore the mode of action (MoA) by which sugammadex interferes with coagulation. The effect of sugammadex on various steps in the coagulation cascade including thrombin generation, factor Xa activity, and factor Xa generation was explored in human plasma. Sugammadex did not affect a conventional thrombin generation test (TGT), while it prolongs activated partial thromboplastin time (APTT) and prothrombin time (PT). However, a customized TGT with PT reagent revealed sugammadex effects. In addition, sugammadex prolonged a one-step prothrombinase-induced clotting time (PiCT) using human factor Xa. Furthermore, sugammadex interfered with factor Xa generation induced by an intrinsic and not by an extrinsic activator, nor by Russell's viper venom factor X (RVV-X). Adapted, rather than standard, experiments show that sugammadex is likely to decrease factor Xa activity in the common pathway and activation of factor X specifically in the intrinsic pathway.

Andexanet versus prothrombin complex concentrates: Differences in reversal of factor Xa inhibitors in in vitro thrombin generation

BackgroundAndexanet alfa (andexanet) is a modified human factor Xa (FXa) approved for anticoagulation reversal in patients with life‐threatening bleeding treated with rivaroxaban or apixaban. Four‐factor prothrombin complex concentrates (4F‐PCCs) are approved for reversal of vitamin K antagonist–induced anticoagulation but not FXa inhibitors. The mechanism and effectiveness of 4F‐PCCs for FXa inhibitor reversal are unclear. ObjectiveTo investigate the mechanism and impact of 4F‐PCCs on reversal of rivaroxaban and apixaban in vitro compared to andexanet. MethodsThe effect of 4F‐PCCs (or individual factors) on tissue factor‐initiated thrombin generation (TF‐TG) was evaluated in human plasma, with or without rivaroxaban or apixaban, and compared with andexanet under the same conditions. ResultsIn the TF‐TG assay, 4F‐PCC completely reversed warfarin anticoagulation. Andexanet normalized TF‐TG over a wide range of apixaban and rivaroxaban concentrations tested (19‐2000 ng/mL). However, 4F‐PCC (or individual factors) was unable to normalize endogenous thrombin potential (ETP) or peak thrombin (Peak) in the presence of apixaban or rivaroxaban (75‐500 ng/mL). TF‐TG was only normalized by 4F‐PCC at inhibitor concentrations <75 ng/mL (ETP) or <37.5 ng/mL (Peak). These data can be explained by the estimated thresholds of FXa activity required to support normal TF‐TG based on the inhibitor:FXa ratios and levels of uninhibited FXa. The data are consistent with healthy volunteer studies where TF‐TG is not normalized until inhibitor levels are substantially decreased. ConclusionsBoth the theoretical calculations and experimental data demonstrated that 4F‐PCCs are only able to normalize TG over a low and narrow range of FXa inhibitor concentrations (<75 ng/mL).

Synthesis, crystal structure, Hirshfeld surface analysis, MEP study and mol-ecular docking of N-{3-[(4-meth-oxy-phen-yl)carbamo-yl]phen-yl}-3-nitro-benzamide as a promising inhibitor of hfXa.

The title compound, C21H17N3O5, consists of three rings, A, B and C, linked by amide bonds with the benzene rings A and C being inclined to the mean plane of the central benzene ring B by 2.99 (18) and 4.57 (18)°, respectively. In the crystal, mol-ecules are linked via N-H⋯O and C-H⋯O hydrogen bonds, forming fused R 2 2(18), R 3 4(30), R 4 4(38) rings running along [0] and R 3 3(37) and R 3 3(15) rings along [001]. Hirshfeld analysis was undertaken to study the inter-molecular contacts in the crystal, showing that the most significant contacts are H⋯O/O⋯H (30.5%), H⋯C/C⋯H (28.2%) and H⋯H (29.0%). Two zones with positive (50.98 and 42.92 kcal mol-1) potentials and two zones with negative (-42.22 and -34.63 kcal mol-1) potentials promote the N-H⋯O inter-actions in the crystal. An evaluation of the mol-ecular coupling of the title compound and the protein with enzymatic properties known as human coagulation factor Xa (hfXa) showed the potential for coupling in three arrangements with a similar minimum binding energy, which differs by approximately 3 kcal mol-1 from the value for the mol-ecule Apixaban, which was used as a positive control inhibitor. This suggests the title compound exhibits inhibitory activity.

Potential Therapeutic Roles for Direct Factor Xa Inhibitors in Coronavirus Infections.

Human factor Xa (FXa) is a serine protease of the common coagulation pathway. FXa is known to activate prothrombin to thrombin, which eventually leads to the formation of cross-linked blood clots. While this process is important in maintaining hemostasis, excessive thrombin generation results in a host of thrombotic conditions. FXa has also been linked to inflammation via protease-activated receptors. Together, coagulopathy and inflammation have been implicated in the pathogenesis of viral infections, including the current coronavirus pandemic. Direct FXa inhibitors have been shown to possess anti-inflammatory and antiviral effects, in addition to their established anticoagulant activity. This review summarizes the pharmacological activities of direct FXa inhibitors, their pharmacokinetics, potential drug–drug interactions and adverse effects, and the details of clinical trials involving direct FXa inhibitors in coronavirus disease 2019 (COVID-19) patients.