Full Study Report of Andexanet Alfa for Bleeding Associated with Factor Xa Inhibitors.

BACKGROUND: Andexanet alfa is a modified recombinant inactive form of human factor Xa developed for reversal of factor Xa inhibitors. METHODS: We evaluated 352 patients who had acute major bleeding within 18 hours after administration of a factor Xa inhibitor. The patients received a bolus of andexanet, followed by a 2-hour infusion. The coprimary outcomes were the percent change in anti-factor Xa activity after andexanet treatment and the percentage of patients with excellent or good hemostatic efficacy at 12 hours after the end of the infusion, with hemostatic efficacy adjudicated on the basis of prespecified criteria. Efficacy was assessed in the subgroup of patients with confirmed major bleeding and baseline anti-factor Xa activity of at least 75 ng per milliliter (or ≥0.25 IU per milliliter for those receiving enoxaparin). RESULTS: Patients had a mean age of 77 years, and most had substantial cardiovascular disease. Bleeding was predominantly intracranial (in 227 patients [64%]) or gastrointestinal (in 90 patients [26%]). In patients who had received apixaban, the median anti-factor Xa activity decreased from 149.7 ng per milliliter at baseline to 11.1 ng per milliliter after the andexanet bolus (92% reduction; 95% confidence interval [CI], 91 to 93); in patients who had received rivaroxaban, the median value decreased from 211.8 ng per milliliter to 14.2 ng per milliliter (92% reduction; 95% CI, 88 to 94). Excellent or good hemostasis occurred in 204 of 249 patients (82%) who could be evaluated. Within 30 days, death occurred in 49 patients (14%) and a thrombotic event in 34 (10%). Reduction in anti-factor Xa activity was not predictive of hemostatic efficacy overall but was modestly predictive in patients with intracranial hemorrhage. CONCLUSIONS: In patients with acute major bleeding associated with the use of a factor Xa inhibitor, treatment with andexanet markedly reduced anti-factor Xa activity, and 82% of patients had excellent or good hemostatic efficacy at 12 hours, as adjudicated according to prespecified criteria. (Funded by Portola Pharmaceuticals; ANNEXA-4 ClinicalTrials.gov number, NCT02329327.).

Hemostatic Efficacy and Anti-FXa (Factor Xa) Reversal With Andexanet Alfa in Intracranial Hemorrhage: ANNEXA-4 Substudy.

Background and Purpose: Andexanet alfa is a recombinant modified human FXa (factor Xa) developed to reverse FXa inhibition from anticoagulants. Hemostatic efficacy and reversal of anti-FXa activity with andexanet were assessed in patients from the ANNEXA-4 study (Andexanet Alfa, a Novel Antidote to the Anticoagulation Effects of FXa Inhibitors) with intracranial hemorrhage (ICrH). Methods: ANNEXA-4 was a single-arm study evaluating andexanet in patients presenting with major bleeding ≤18 hours after taking an FXa inhibitor. Patients received a bolus plus 2-hour infusion of andexanet. Brain imaging in patients with ICrH was performed at baseline and at 1 and 12 hours postandexanet infusion. Coprimary efficacy outcomes were change in anti-FXa activity and hemostatic efficacy at 12 hours (excellent/good efficacy defined as ≤35% increase in hemorrhage volume/thickness). Safety outcomes included occurrence of thrombotic events and death at 30 days. Results: A total of 227 patients with ICrH were included in the safety population (51.5% male; mean age 79.3 years) and 171 in the efficacy population (99 spontaneous and 72 traumatic bleeds). In efficacy evaluable patients, excellent/good hemostasis 12 hours postandexanet occurred in 77 out of 98 (78.6%) and in 58 out of 70 (82.9%) patients with spontaneous and traumatic bleeding, respectively. In the subanalysis by FXa inhibitor treatment group in the efficacy population, median of percent change in anti-FXa from baseline to nadir showed a decrease of 93.8% for apixaban-treated patients (n=99) and by 92.6% for rivaroxaban-treated patients (n=59). Within 30 days, death occurred in 34 out of 227 (15.0%) patients and thrombotic events occurred in 21 out of 227 (9.3%) patients (safety population). Conclusions: Andexanet reduced anti-FXa activity in FXa inhibitor-treated patients with ICrH, with a high rate of hemostatic efficacy. Andexanet may substantially benefit patients with ICrH, the most serious complication of anticoagulation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02329327.

Andexanet Alfa for Acute Major Bleeding Associated with Factor Xa Inhibitors.

BACKGROUND: Andexanet alfa (andexanet) is a recombinant modified human factor Xa decoy protein that has been shown to reverse the inhibition of factor Xa in healthy volunteers. METHODS: In this multicenter, prospective, open-label, single-group study, we evaluated 67 patients who had acute major bleeding within 18 hours after the administration of a factor Xa inhibitor. The patients all received a bolus of andexanet followed by a 2-hour infusion of the drug. Patients were evaluated for changes in measures of anti-factor Xa activity and were assessed for clinical hemostatic efficacy during a 12-hour period. All the patients were subsequently followed for 30 days. The efficacy population of 47 patients had a baseline value for anti-factor Xa activity of at least 75 ng per milliliter (or ≥0.5 IU per milliliter for those receiving enoxaparin) and had confirmed bleeding severity at adjudication. RESULTS: The mean age of the patients was 77 years; most of the patients had substantial cardiovascular disease. Bleeding was predominantly gastrointestinal or intracranial. The mean (±SD) time from emergency department presentation to the administration of the andexanet bolus was 4.8±1.8 hours. After the bolus administration, the median anti-factor Xa activity decreased by 89% (95% confidence interval [CI], 58 to 94) from baseline among patients receiving rivaroxaban and by 93% (95% CI, 87 to 94) among patients receiving apixaban. These levels remained similar during the 2-hour infusion. Four hours after the end of the infusion, there was a relative decrease from baseline of 39% in the measure of anti-factor Xa activity among patients receiving rivaroxaban and of 30% among those receiving apixaban. Twelve hours after the andexanet infusion, clinical hemostasis was adjudicated as excellent or good in 37 of 47 patients in the efficacy analysis (79%; 95% CI, 64 to 89). Thrombotic events occurred in 12 of 67 patients (18%) during the 30-day follow-up. CONCLUSIONS: On the basis of a descriptive preliminary analysis, an initial bolus and subsequent 2-hour infusion of andexanet substantially reduced anti-factor Xa activity in patients with acute major bleeding associated with factor Xa inhibitors, with effective hemostasis occurring in 79%. (Funded by Portola Pharmaceuticals; ANNEXA-4 ClinicalTrials.gov number, NCT02329327 .).

Transient or extended reversal of apixaban anticoagulation by andexanet alfa is equally effective in a porcine polytrauma model.

BACKGROUND: Andexanet alfa (andexanet) reverses the anticoagulant effects of factor Xa inhibitors, but it has not been assessed in clinical studies for apixaban reversal in trauma. This study evaluated andexanet for reversing apixaban anticoagulation in a porcine polytrauma model. METHODS: Oral apixaban (20 mg q.d., n=21) or placebo (n=7; sham group) was administered to male pigs for 4 days before blunt liver injury and bi-lateral femur fracture. After trauma, animals were randomised 1:1:1 to a single andexanet bolus (1000 mg), a bolus (1000 mg) plus infusion (1200 mg over 2 h), or vehicle (control). Haemodynamic and coagulation variables were monitored for 5 h or until death. The primary endpoint was blood loss. RESULTS: Mean blood loss in sham animals was 472 (standard deviation, 58) ml 12 min after injury and 658 (98) ml at 300 min, with 100% survival. Anticoagulation with apixaban significantly increased blood loss 12 min after injury [888 (133) ml, P<0.01]. Controls exhibited total blood loss of 3403 (766) ml, with 100% mortality. Andexanet bolus or bolus plus infusion significantly reduced blood loss to 1264 (205) and 1202 (95) ml, respectively), and increased survival to 100%. Haemodynamic parameters and markers of shock recovered to pre-trauma levels in andexanet-treated animals. CONCLUSION: Andexanet effectively reversed apixaban anticoagulation and reduced blood loss induced by severe trauma. Andexanet bolus alone had a similar impact on survival and blood loss as bolus plus infusion. Therefore, a 2 h andexanet infusion after the bolus may not be necessary to restore normal haemostatic mechanisms.

Andexanet Alfa for the Reversal of Factor Xa Inhibitor Activity.

BACKGROUND: Bleeding is a complication of treatment with factor Xa inhibitors, but there are no specific agents for the reversal of the effects of these drugs. Andexanet is designed to reverse the anticoagulant effects of factor Xa inhibitors. METHODS: Healthy older volunteers were given 5 mg of apixaban twice daily or 20 mg of rivaroxaban daily. For each factor Xa inhibitor, a two-part randomized placebo-controlled study was conducted to evaluate andexanet administered as a bolus or as a bolus plus a 2-hour infusion. The primary outcome was the mean percent change in anti-factor Xa activity, which is a measure of factor Xa inhibition by the anticoagulant. RESULTS: Among the apixaban-treated participants, anti-factor Xa activity was reduced by 94% among those who received an andexanet bolus (24 participants), as compared with 21% among those who received placebo (9 participants) (P<0.001), and unbound apixaban concentration was reduced by 9.3 ng per milliliter versus 1.9 ng per milliliter (P<0.001); thrombin generation was fully restored in 100% versus 11% of the participants (P<0.001) within 2 to 5 minutes. Among the rivaroxaban-treated participants, anti-factor Xa activity was reduced by 92% among those who received an andexanet bolus (27 participants), as compared with 18% among those who received placebo (14 participants) (P<0.001), and unbound rivaroxaban concentration was reduced by 23.4 ng per milliliter versus 4.2 ng per milliliter (P<0.001); thrombin generation was fully restored in 96% versus 7% of the participants (P<0.001). These effects were sustained when andexanet was administered as a bolus plus an infusion. In a subgroup of participants, transient increases in levels of d-dimer and prothrombin fragments 1 and 2 were observed, which resolved within 24 to 72 hours. No serious adverse or thrombotic events were reported. CONCLUSIONS: Andexanet reversed the anticoagulant activity of apixaban and rivaroxaban in older healthy participants within minutes after administration and for the duration of infusion, without evidence of clinical toxic effects. (Funded by Portola Pharmaceuticals and others; ANNEXA-A and ANNEXA-R ClinicalTrials.gov numbers, NCT02207725 and NCT02220725.).

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice.

OBJECTIVE: Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice lacking CDGI or P2Y12 in hematopoietic cells. APPROACH AND RESULTS: Lethally irradiated Ldlr(-/-) mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1(-/-) (cdgI(-/-)), p2y12(-/-), or cdgI(-/-)p2y12(-/-) (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr(-/-) chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr(-/-)/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/p2y12(-/-) chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/ p2y12(-/-) chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr(-/-) chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr(-/-)/DKO chimeras when compared with chimeras lacking only CDGI. CONCLUSIONS: Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr(-/-) mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor.

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin.

Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.

Rap1-Rac1 circuits potentiate platelet activation.

OBJECTIVE: The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. METHODS AND RESULTS: We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI- and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. CONCLUSIONS: We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI- and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP.

The effects of ADP on the biology of dendritic cells have been studied much less than those of ATP or adenosine. In this study, we showed that adenosine-5'-O-(2-thiodiphosphate) (ADPßS) induced intracellular Ca(2+) transients in murine dendritic cells (DCs). This effect was abolished by AR-C69931MX, a dual P2Y(12) and P2Y(13) receptor antagonist. RT-PCR experiments revealed the expression of both P2Y(12) and P2Y(13) mRNA in DCs. The Ca(2+) response to ADPßS was maintained in P2Y(13)-deficient DCs, whereas it was abolished completely in P2Y(12)(-/-) DCs. ADPßS stimulated FITC-dextran and OVA capture in murine DCs through macropinocytosis, and this effect was abolished in P2Y(12)(-/-) DCs. ADPßS had a similar effect on FITC-dextran uptake by human monocyte-derived DCs. OVA loading in the presence of ADPßS increased the capacity of DCs to stimulate OVA-specific T cells, whereas ADPßS had no effect on the ability of DCs to stimulate allogeneic T cells. Moreover, after immunization against OVA, the serum level of anti-OVA IgG1 was significantly lower in P2Y(12)(-/-) mice than that in wild-type controls. In conclusion, we have shown that the P2Y(12) receptor is expressed in murine DCs and that its activation increased Ag endocytosis by DCs with subsequent enhancement of specific T cell activation.

Development of a perfusion chamber assay to study in real time the kinetics of thrombosis and the antithrombotic characteristics of antiplatelet drugs.

BACKGROUND: Arterial thrombosis triggered by vascular injury is a balance between thrombus growth and thrombus fragmentation (dethrombosis). Unbalance towards thrombus growth can lead to vascular occlusion, downstream ischemia and tissue damage.Here we describe the development of a simple methodology that allows for continuous real time monitoring and quantification of both processes during perfusion of human blood under arterial shear rate conditions. Using this methodology, we have studied the effects of antiplatelet agents targeting COX-1 (aspirin), P2Y12 (2-MeSAMP, clopidogrel), GP IIb-IIIa (eptifibatide) and their combinations on the kinetics of thrombosis over time. RESULTS: Untreated samples of blood perfused over type III collagen at arterial rates of shear promoted the growth of stable thrombi. Modulation by eptifibatide affected thrombus growth, while that mediated by 2-MeSAMP and aspirin affected thrombus stability. Using this technique, we confirmed the primacy of continuous signaling by the ADP autocrine loop acting on P2Y12 in the maintenance of thrombus stability. Analysis of the kinetics of thrombosis revealed that continuous and prolonged analysis of thrombosis is required to capture the role of platelet signaling pathways in their entirety. Furthermore, studies evaluating the thrombotic profiles of 20 healthy volunteers treated with aspirin, clopidogrel or their combination indicated that while three individuals did not benefits from either aspirin or clopidogrel treatments, all individuals displayed marked destabilization profiles when treated with the combination regimen. CONCLUSIONS: These results show the utility of a simple perfusion chamber technology to assess in real time the activity of antiplatelet drugs and their combinations. It offers the opportunity to perform pharmacodynamic monitoring of arterial thrombosis in clinical trials and to investigate novel strategies directed at inhibiting thrombus stability in the management of cardiovascular disease.

A clopidogrel-insensitive inducible pool of P2Y12 receptors contributes to thrombus formation: inhibition by elinogrel, a direct-acting, reversible P2Y12 antagonist.

It is known that hepatic metabolism limits the antiaggregatory activity of clopidogrel and, as a consequence, its clinical benefits. In this study, we investigated whether other factors exist that could account for clopidogrel's suboptimal antithrombotic activity. Using an in vivo murine FeCl(3) thrombosis model coupled with intravital microscopy, we found that at equivalent, maximal levels of inhibition of ADP-induced platelet aggregation, clopidogrel (50 mg/kg p.o.) failed to reproduce the phenotype associated with P2Y(12) deficiency. However, elinogrel (60 mg/kg p.o.), a direct-acting reversible P2Y(12) antagonist, achieved maximal levels of inhibition in vivo, and its administration (1 mg/kg i.v.) abolished residual thrombosis associated with clopidogrel dosing. Because elinogrel is constantly present in the plasma, whereas the active metabolite of clopidogrel exists for ∼2 h, we evaluated whether an intracellular pool of P2Y(12) exists that would be inaccessible to clopidogrel and contribute to its limited antithrombotic activity. Using saturation [(3)H]2-(methylthio)ADP ([(3)H]2MeSADP) binding studies, we first demonstrated that platelet stimulation with thrombin and convulxin (mouse) and thrombin receptor activating peptide (TRAP) (human) significantly increased surface expression of P2Y(12) relative to that of resting platelets. We next found that clopidogrel dose-dependently inhibited ADP-induced aggregation, signaling (cAMP), and surface P2Y(12) on resting mouse platelets, achieving complete inhibition at the highest dose (50 mg/kg), but failed to block this inducible pool. Thus, an inducible pool of P2Y(12) exists on platelets that can be exposed upon platelet activation by strong agonists. This inducible pool is not blocked completely by clopidogrel, contributes to thrombosis in vivo, and can be blocked by elinogrel.

Thienopyridines, but not elinogrel, result in off-target effects at the vessel wall that contribute to bleeding.

Clinical studies with clopidogrel or prasugrel show that although increased inhibition of P2Y(12) and platelet function improves efficacy, bleeding is also increased. Other preclinical and clinical studies have suggested a greater therapeutic index (TI) with reversible inhibitors and disproportionate effects of thienopyridines on bleeding at high doses. We used multiple in vivo (FeCl(3)-induced arterial thrombosis in mesenteric arteries, blood loss after tail transsection, and platelet deposition and wound closure time in a micropuncture model in mesenteric veins) and ex vivo (light transmittance aggregometry, prothrombin time, and activated partial thromboplastin time) mouse models to 1) compare the TI of clopidogrel, prasugrel, and elinogrel, a reversible, competitive antagonist, with that in P2Y(12)(-/-) mice and 2) determine whether the bleeding consequences of the thienopyridines are attributed only to the inhibition of P2Y(12). Data indicated greater (elinogrel) and decreased (thienopyridines) TI compared with that in P2Y(12)(-/-) mice. The impaired TI associated with the thienopyridines was not attributed to non-P2Y(12) activities on platelet function or coagulation but was related to a direct effect at the vessel wall (inhibition of vascular tone). Further analysis showed that the prasugrel off-target effect was dose- and time-dependent and of a reversible nature. In conclusion, the TI of thienopyridines in the mouse may be decreased by P2Y(12)-independent off-target effects at the vessel wall, whereas that of elinogrel may be enhanced by the reversible, competitive nature of the antiplatelet agent.

Measuring residual anti-Xa activity of direct factor Xa inhibitors after reversal with andexanet alfa.

INTRODUCTION: Andexanet alfa (AnXa) was developed for anticoagulant effect reversal of direct factor Xa inhibitors (DXaI) (apixaban, rivaroxaban, edoxaban) in emergency situations. Regular anti-Xa assays are not suitable to evaluate anti-Xa activity after AnXa administration because of the high sample dilution resulting in the AnXa-DXaI dissociation which gives inaccurately high DXaI measured concentrations. This study aimed at developing dedicated STA-Liquid anti-Xa test set-ups for accurately measuring DXaI after reversal with AnXa. METHODS: Modified anti-Xa test set-ups, with reduced sample dilution, were developed to overcome regular assays limitations and to improve measured accuracy with results comparable to Portola microplate reference method used in clinical studies. Both regular and optimized assays were used to measure DXaI concentration in AnXa-containing samples. Quality controls, normal pooled plasma spiked with five DXaI and three AnXa concentrations, samples from DXaI-treated patients spiked with AnXa and ex vivo healthy volunteers having received both DXaI and AnXa were used. RESULTS: The lower limit of quantitation of optimized anti-Xa assays was <10 ng/mL with CVs ≤10%. DXaI samples containing 300 ng/mL and 1 µmol/L AnXa resulted in DXaI residual concentrations of 29-72 ng/mL depending on the DXaI (76%-90% reversal), compared to 20-28 ng/mL with reference method (92%-94% reversal) and 135-165 ng/mL with regular assays (about 50% reversal). CONCLUSION: Modified test set-ups are automated alternative to reference method with improved precision and reproducibility. They can be run in all laboratories where regular anti-Xa assays are performed using commercially available reagents.

G-protein-coupled receptors as signaling targets for antiplatelet therapy.

Platelet G protein-coupled receptors (GPCRs) initiate and reinforce platelet activation and thrombus formation. The clinical utility of antagonists of the P2Y(12) receptor for ADP suggests that other GPCRs and their intracellular signaling pathways may represent viable targets for novel antiplatelet agents. For example, thrombin stimulation of platelets is mediated by 2 protease-activated receptors (PARs), PAR-1 and PAR-4. Signaling downstream of PAR-1 or PAR-4 activates phospholipase C and protein kinase C and causes autoamplification by production of thromboxane A(2), release of ADP, and generation of more thrombin. In addition to ADP receptors, thrombin and thromboxane A(2) receptors and their downstream effectors-including phosphoinositol-3 kinase, Rap1b, talin, and kindlin-are promising targets for new antiplatelet agents. The mechanistic rationale and available clinical data for drugs targeting disruption of these signaling pathways are discussed. The identification and development of new agents directed against specific platelet signaling pathways may offer an advantage in preventing thrombotic events while minimizing bleeding risk.

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

BACKGROUND: Andexanet 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. OBJECTIVE: To investigate the mechanism and impact of 4F-PCCs on reversal of rivaroxaban and apixaban in vitro compared to andexanet. METHODS: The 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. RESULTS: In 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. CONCLUSIONS: Both 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).

A phase 2 PK/PD study of andexanet alfa for reversal of rivaroxaban and edoxaban anticoagulation in healthy volunteers.

As with any anticoagulant, factor Xa (FXa) inhibitors are associated with a risk of major bleeding. Andexanet alfa is a recombinant modified human FXa lacking enzymatic activity, developed for reversal of FXa inhibitor-induced anticoagulation. In two phase 2, randomized, double-blind, placebo-controlled, single-center studies, different regimens of andexanet alfa were administered to healthy volunteers after therapeutic anticoagulation with rivaroxaban or edoxaban, and multiple anticoagulation reversal and safety end points were evaluated. Andexanet alfa rapidly and effectively reversed anticoagulation with both rivaroxaban and edoxaban. Within 2 minutes after bolus, anti-FXa activity decreased significantly, with maximum decreases of ≈93% (P < .05) and ≈82% (P < .05), respectively, compared with placebo. The stoichiometric ratios of andexanet alfa:total anticoagulant at maximum reversal of anti-FXa activity ranged from 1:1 to 1.3:1 for rivaroxaban and 1.41:1 to 2.58:1 for edoxaban. Sustained normalization of thrombin generation for ≈2 hours and sustained decrease in unbound anticoagulant (maximum ≈80%) for up to ≈4 hours following completion of andexanet alfa administration, compared with placebo, were observed when andexanet was administered as a bolus or as a bolus followed by continuous infusion. Andexanet alfa was well tolerated, and there were no serious adverse events or thrombotic events. Andexanet alfa has been approved in the United States and Europe for reversal of anticoagulation in patients treated with rivaroxaban or apixaban who experience life-threatening or uncontrolled bleeding. These studies were registered with clinicaltrials.gov (#NCT03578146 and #NCT03551743).