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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

Outcomes following andexanet alfa reversal of factor Xa inhibitors in patients requiring urgent or emergent invasive procedures are lacking. This study aimed to describe efficacy and safety outcomes following andexanet alfa administration within 24 h of an invasive procedure.This single-center, observational, retrospective study included patients who received andexanet alfa within 24 h of an invasive or surgical procedure. The primary outcome was hemostatic efficacy graded as excellent, good, or poor using similar definitions to the ANNEXA-4 criteria. Secondary outcomes included hospital discharge disposition, intensive care unit (ICU) and hospital length of stay, 30-day mortality, 30-day thromboischemic event rates, and serum coagulation assay changes pre- and postreversal.Forty-four patients met inclusion criteria; of these, 27 (62.8%) received apixaban and 16 (37.2%) were treated with rivaroxaban prior to admission. The indications for reversal were categorized as intracranial (n = 20 [45.5%]) or extracranial (n = 24 [54.5%]) sites. Majority of patients required emergent operative procedures (18 [40.9%]), followed by invasive device placement (10 [22.7%]) or arterial embolization (9 [20.5%]). Thirty-eight (86.4%) patients were able to be adequately graded for hemostatic efficacy. Overall, 30 (78.9%) patients achieved excellent or good hemostasis within 24 h after periprocedural administration of andexanet alfa (19 [82.6%] apixaban vs. 11 [78.6%] rivaroxaban; 12 [80.0%] intracranial events vs. 18 [78.3%] extracranial events). Discharge disposition was most often to a short- or long-term care facilities (27 [61.4%]). Thirty-day mortality and thromboischemic complications occurred in 15 (34.1%) and 12 (27.3%) patients, respectively. Prothrombin time and antifactor Xa assay results were significantly decreased after andexanet alfa administration (p 0.05) while thromboelastogram assay values (reaction time, kinetic time, and activated clotting time) showed nonsignificant changes pre- versus postreversal.Andexanet alfa may be used for urgent or emergent reversal of apixaban and rivaroxaban peri-procedurally with promising hemostatic outcomes. Further prospective, comparative clinical research is warranted.

Blood coagulation is an intricate process, and it requires precise control of the activities of pro- and anticoagulant factors and sensitive signaling systems to monitor and respond to blood vessel insults. These requirements are fulfilled by phosphatidylserine, a relatively miniscule-sized lipid molecule amid the myriad of large coagulation proteins. This review limelight the role of platelet membrane phosphatidylserine (PS) in regulating a key enzymatic reaction of blood coagulation; conversion of factor X to factor Xa by the enzyme factor IXa and its cofactor factor VIIIa. PS is normally located on the inner leaflet of the resting platelet membrane but appears on the outer leaflet surface of the membrane surface after an injury happens. Human platelet activation leads to exposure of buried PS molecules on the surface of the platelet-derived membranes and the exposed PS binds to discrete and specific sites on factors IXa and VIIIa. PS binding to these sites allosterically regulates both factors IXa and VIIIa. The exposure of PS and its binding to factors IXa/VIIIa is a vital step during clotting. Insufficient exposure or a defective binding of PS to these clotting proteins is responsible for various hematologic diseases which are discussed in this review.

Patients experiencing an intracranial hemorrhage (ICH) on oral anticoagulants often require rapid reversal. This study evaluated patients taking factor Xa inhibitors or warfarin that received reversal with 4-factor prothrombin complex concentrate (4F-PCC) for an ICH. The objective of the study was to determine if the efficacy of 4F-PCC for the reversal of factor Xa inhibitors is noninferior to its use in warfarin reversal in patients with ICH.This was a retrospective, single center, noninferiority trial. Patients presenting to the emergency department with ICH were divided into two cohorts: those taking factor Xa inhibitors versus those taking warfarin. In each cohort, patients received anticoagulation reversal with weight-based 4F-PCC. The primary endpoint was hemostatic efficacy defined as ≤20% expansion in hematoma volume on repeat computed tomography imaging. A pre-specified noninferiority margin of -10% was selected to evaluate the difference between groups for the primary endpoint.A total of 221 patients were included in the study (factor Xa inhibitors, n = 87; warfarin, n = 134). Effective hemostasis was achieved in 70 patients (81%) on factor Xa inhibitors compared to 111 patients (83%) on warfarin, (-2.4% difference, [95% confidence interval, -12.87 to 8.12]; p = 0.654). There was no statistically significant difference between groups with regards to the primary outcome; however, the use of 4F-PCC in factor Xa inhibitor reversal was not noninferior when compared to 4F-PCC use for warfarin reversal. Hospital length of stay and discharge disposition were similar between cohorts.The efficacy of 4F-PCC in reversing factor Xa inhibitor-related ICH compared to warfarin-related ICH was not significantly different between groups; however, these results did not prove noninferiority. Further study is warranted to delineate 4F-PCC's role in reversing factor Xa inhibitors in patients with ICH.

The prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in the opposite order. For the less favorable cleavage site at Arg320 to be cleaved first, it is thought that prothrombin docks on fVa in a way that presents Arg320 and hides Arg271 from the active site of fXa. Based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C, we modeled an initial prothrombin docking mode, which involved an interaction with discrete portions of the A1 and A2 domains of fV and the loop connecting the 2 domains, known as the a1-loop. We interrogated the proposed interface by site-directed PEGylation and by swapping the a1-loop in pseutarin C with that of human fV and fVIII and measuring the effect on rate and pathway of thrombin generation. PEGylation of residues within our proposed binding site greatly reduced the rate of thrombin generation, without affecting the pathway, whereas those outside the proposed interface had no effect. PEGylation of residues within the a1-loop also reduced the rate of thrombin generation. The sequence of the a1-loop was found to play a critical role in prothrombin binding and in the presentation of Arg320 for initial cleavage.

The ongoing controversy regarding optimal reversal agent for factor Xa-inhibitors is mainly due to lack of comparative data of andexanet alfa (AA) to 4-factor prothrombin complex concentrate (4F-PCC), institutional formulary restrictions, and navigation of clinical scenarios involving patients clinically worsen despite initial reversal efforts. The combination use of 4F-PCC and AA has not been evaluated in clinical trials and the outcomes of such patients with FXA-inhibitor associated intracranial hemorrhage (ICH) are unknown. A total of five patients, including four outside hospital transfers, received 4F-PCC prior to AA for FXa-inhibitor associated ICH (n = 3 apixaban, n = 2 rivaroxaban; n = 4 ICH, n = 1 TBI). The doses of 4F-PCC ranged from 25 to 60 units/kg and were administered within a range of 1.5-4.2 h prior to AA. One patient required surgical intervention with craniotomy and three patients underwent external ventricular drain placement. Two of the five patients developed an ischemic or thromboembolic complication within one week from 4F-PCC and AA administration. This case series discusses multiple unique patient cases in which 4F-PCC and AA were both administered for FXa-inhibitor associated ICH. The results highlight the potentially increased thrombotic risk associated with combination use. Ongoing post-marketing data collection of real patient case scenarios are essential to the establishment of consensus guidelines on how to prioritize initial reversal efforts and manage these patients during the course of their bleed.

There is limited information directly comparing andexanet alfa (AA) versus four-factor prothrombin complex concentrate (4F-PCC) in intracranial hemorrhage (ICH) on apixaban or rivaroxaban.The objective of this study was to compare the effectiveness and safety of AA versus 4F-PCC in ICH on apixaban or rivaroxaban.This retrospective, matched, cohort analysis was conducted at a single healthcare system. Patients were matched based on baseline ICH volume. The primary outcome was good or excellent ICH hemostasis, which was defined as a 35% or less increase in ICH volume within 24 h following AA or 4F-PCC administration. The secondary outcome was thrombotic events within 14 days following AA or 4F-PCC administration.In total, 26 AA and 26 4F-PCC patients were included in this matched cohort analysis. Both groups had comparable rates of good or excellent ICH hemostasis (AA: 92.3% vs. 4F-PCC: 88.5%, p = 1.000). Thrombotic events within 14-days were not significantly different (AA: 26.9% vs. 4F-PCC: 11.5%, p = 0.159).This study found no significant differences in good or excellent ICH hemostasis within 24-h or new thrombotic events within 14-days in a cohort given AA or 4F-PCC for ICH while on apixaban or rivaroxaban. However, this single-center analysis is underpowered due to sample size constraints, therefore further high-quality research comparing AA safety and effectiveness versus 4F-PCC is needed.

Factor V-short (FV756-1458) is a natural splice variant functioning in synergy with protein S as tissue factor pathway inhibitor alpha (TFPIα)-cofactor in inhibition of factor Xa (FXa). An exposed acid region (AR2; 1493-1537) in the B domain binds TFPIα. The preAR2 (1458-1492) is crucial for the synergistic TFPIα-cofactor activity between FV-short and protein S and for assembly of a trimolecular FXa-inhibitory complex among FV-short, protein S, and TFPIα.To identify which part of preAR2 is required for the synergistic TFPIα-cofactor activity between FV-short and protein S.A FXa-inhibition assay was used to test the synergistic TFPIα cofactor activity between protein S and new FV-short variants FV709-1476, FV712-1478, FV712-1481, FV712-1484, FV712-1487, and FV712-1490. A microtiter-based assay analyzed binding among FV-short variants, protein S, and TFPIα.FV709-1476, FV712-1478, and FV712-1481 were fully active as synergistic TFPIα cofactors with protein S; FV712-1484 showed intermediate activity; and FV712-1487 and FV712-1490 were inactive. TFPIα interacted with all variants in the absence of protein S but FV712-1478 and FV712-1481 bound TFPIα with highest affinity. None of the FV-short variants bound directly to protein S in the absence of TFPIα. In the presence of TFPIα, efficient cooperative binding was demonstrated between protein S, TFPIα, and FV709-1476, FV712-1478, or FV712-1481. In contrast, no cooperativity among TFPIα, protein S, and FV712-1484, FV712-1487, or FV712-1490 was seen.A short hydrophobic patch in preAR2 (PLVIVG, 1481-1486) in FV-short is crucial for the synergistic TFPIα-cofactor activity between FV-short and protein S and for the assembly of a trimolecular FXa-inhibitory complex among FV-short, protein S, and TFPIα.

Existing research recommends either andexanet alfa (AA) or four-factor prothrombin complex concentrate (4F-PCC) as an antidote for major bleeding events due to apixaban or rivaroxaban. Currently, there is limited published research that directly compares the risks and benefits of the two agents in patients with oral factor Xa inhibitor related traumatic and spontaneous intracerebral hemorrhages. Additional head-to-head data is needed to support favoring either AA or 4F-PCC when it comes to efficacy, safety, and cost.A retrospective chart review was conducted to assess patients admitted to a multi-center healthcare system and a stand-alone teaching hospital in central Florida from June 2016 to December 2020. Patients included in the study were at least 18 years of age, taking apixaban or rivaroxaban prior to admission, had radiographical evidence of an intracranial hemorrhage, and received either AA or 4F-PCC as a reversal agent. The primary outcome analyzed was the level of excellent hemostasis achieved, based on a standardized rating system for effective hemostasis defined by the International Society of Thrombosis and Hemostasis (ISTH), after administration of AA or 4F-PCC. Secondary outcomes analyzed included changes in the initial hemorrhage volume as reported on computed tomography (CT) scan and at 12 to 24 h post treatment, rate of thromboembolic events, rate of inpatient mortality, and total cost of treatment after AA or 4F-PCC administration.A total of 109 patients were included in the study with 47 in the AA group (43.1%) and 62 in the 4F-PCC group (56.9%). There were no statistically significant differences between AA and 4F-PCC in terms of the primary and secondary outcomes with the exception of total cost of treatment. The level of excellent hemostasis achieved after reversal administration of AA was seen in 27 patients (71.1%) and 41 patients (70.7%) after 4F-PCC administration (p = 1, p adjusted = 0.654 after controlling for age, ICH score, regional mass effect, and midline shift). There was no statistically significant difference in the median percentage change in hemorrhagic volume from baseline to 12-24 h after reversal treatment (0 [-0.17--0.24] vs. 0 [-0.021-0.29], p = 0.439, adjusted p = 0.601) in the AA and 4F-PCC groups, respectively. The total incidence of thromboembolic events (4 [8.5%] vs. 6 [9.7%], p = 1, adjusted p = 0.973) and rate of inpatient mortality was similar between the two groups (16 [34.0%] vs. 13 [21.0%], p = 0.134, adjusted p = 0.283). A statistically significant difference was observed with the total cost of reversal treatment: $23,602 for treatment with AA and $6692 for treatment with 4F-PCC.No statistically significant differences were identified in primary or secondary outcomes between the two agents with the exception of total treatment cost. There is insufficient evidence based on this study to recommend AA over 4F-PCC for patients with intracranial hemorrhages associated with the use of apixaban or rivaroxaban.

PurposeTo manage factor Xa (FXa) inhibitor–associated bleeding, andexanet alfa or 4-factor prothrombin concentrate (4F-PCC) has been used to restore hemostasis. However, literature on the outcomes for patients who received both andexanet alfa and 4F-PCC is limited.SummaryWe report a case series of 5 patients who received andexanet alfa plus 4F-PCC for reversal of FXa inhibitor–associated bleeding. Patients were included in this case series if they received both andexanet alfa and 4F-PCC for reversal of FXa inhibitor–associated bleeding. They were followed to either discharge or death, and in-hospital complications related to concurrent use of andexanet alfa and 4F-PCC were documented. We report an incidence of thromboembolism of 40% (2 of 5 cases) and an in-hospital mortality rate of 60% (3 of 5 cases). Taking these cases together with those in the existing literature, we found a total of 23 reported cases of safety outcomes with andexanet alfa plus 4F-PCC. The overall incidence of thromboembolism was 35% (8 of 23 cases).ConclusionThis case series adds to the limited literature describing the outcomes for patients receiving andexanet alfa plus 4F-PCC. We encourage other institutions to report safety data on administering both agents.

Background Andexanet alfa (andexanet) is approved for specific anticoagulation reversal in patients with life-threatening or uncontrolled bleeding during treatment with rivaroxaban or apixaban. There is limited experience with andexanet in patients with acute bleeding on edoxaban. Methods Patients with acute major bleeding within 18 hours of edoxaban intake were prospectively enrolled. Patients received a bolus and 2-hour follow-on infusion of andexanet. The co-primary efficacy outcomes were change in antifactor Xa activity and the percentage of patients achieving excellent or good hemostasis, 12 hours after andexanet treatment. Efficacy was analyzed in patients with confirmed major bleeding and baseline antifactor Xa activity ≥40 ng/mL. Safety was analyzed in all patients. Results Thirty-six patients (mean age: 82 years, 61.1% male and 91.7% with atrial fibrillation) with acute major bleeding on edoxaban received andexanet. The primary site of bleeding was intracranial in 29 patients (80.6%). In the efficacy population (n = 28), median antifactor Xa activity decreased from 121.1 (interquartile range [IQR]: 70.3–202.4) ng/mL at baseline to 24.0 (IQR: 77.7–83.7) ng/mL at the end of andexanet bolus (median decrease: 68.9%, 95% confidence interval [CI]: 56.1–77.7%). Excellent or good hemostasis at 12 hours was achieved in 78.6% (95% CI: 59.0–91.7%) of patients. Within 30 days, four patients (11.1%) experienced a thrombotic event and four others (11.1%) died. Conclusion In patients with acute major bleeding on edoxaban, andexanet significantly decreased antifactor Xa activity. Hemostatic efficacy was similar to that observed in patients with bleeding on rivaroxaban or apixaban. Thrombotic events occurred at a rate expected in such patients.

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.

Apixaban is used to treat venous thromboembolism (VTE) at 10 mg twice daily (BID) for 7 days, followed by 5 mg BID without dose adjustment, and non-valvular atrial fibrillation (NVAF) at 5 mg BID or 2.5 mg BID with dose adjustment criteria (DAC) including age, body weight, and renal function. The anti-factor Xa activity (AXA), prothrombin time (PT), and activated partial thromboplastin time (APTT) in patients with VTE receiving 10 mg BID of apixaban remains unclear. Twenty-six patients (70.8±15.4 years, 10 males) with VTE receiving 10 mg BID of apixaban were enrolled. The patients were divided into two groups based on whether they met the DAC of NVAF: DAC group (n=8) and non-DAC group (n=18). Trough and peak AXA values, PT, and APTT were measured at 10 mg BID dosage and then at 5 mg BID dosage. Coagulation markers in recipients of 10 mg BID therapy were significantly higher than those of 5 mg BID recipients. A significant and strong positive correlation was observed between AXA and PT at trough and peak times. The AXA values and PT in the DAC group were significantly higher than those in the non-DAC group. No significant inter-group differences were seen in APTT. This study provides the first report of AXA distribution in VTE patients receiving 10 mg BID of apixaban. Our findings indicate that coagulation markers may differ in patients with VTE-prescribed higher doses of apixaban and a DAC may be warranted in such patients.

Objective The optimal intensity of anticoagulation for adult patients supported with extracorporeal membrane oxygenation (ECMO) remains uncertain. The objective of this study was to evaluate the effectiveness and safety of two anticoagulation protocols using conventional (0.3-0.7 IU/ml) versus restricted (0.2-0.5 IU/ml) anti-factor Xa (anti-Xa) targets for the management of unfractionated heparin (UFH) in adult ECMO patients. Methods This retrospective before-after cohort study compared two groups of ECMO patients who received UFH for at least 24-h from March 2016 to May 2019. The primary outcome was the composite rate of major bleeding or thrombotic events per ECMO day. Secondary outcomes included the mean amount of blood products transfused per ECMO day, the proportion of patients who were within the target anti-Xa at 24-h, the time to achieve target anti-Xa, and the number of heparin infusion adjustments to reach target anti-Xa. Results Forty-one patients were included in this analysis (conventional, n = 25; restricted, n = 16). There was no difference in the composite rate of major bleeding or thrombotic events per ECMO day (p = .090). The restricted group had lower rates of packed red blood cells (pRBC) transfusion per ECMO day (mean 1 ± 1 vs 3 ± 2 units, p = .003) and required fewer heparin infusion adjustments to reach the target (p = .007). There was no difference between the groups in the number of patients who achieved target anti-Xa at 24-h (p = .940). Conclusion In adult ECMO patients, anticoagulation with a restricted anti-Xa target was associated with lower pRBC transfusions and did not provoke an excess of thrombotic events.

In silico driven optimization of compound properties related to pharmacokinetics, pharmacodynamics, and safety is a key requirement in modern drug discovery. Nowadays, large and harmonized datasets allow to implement deep neural networks (DNNs) as a framework for leveraging predictive models. Nevertheless, various available model architectures differ in their global applicability and performance in lead optimization projects, such as stability over time and interpretability of the results. Here, we describe and compare the value of established DNN-based methods for the prediction of key ADME property trends and biological activity in an industrial drug discovery environment, represented by microsomal lability, CYP3A4 inhibition and factor Xa inhibition. Three architectures are exemplified, our earlier described multilayer perceptron approach (MLP), graph convolutional network-based models (GCN) and a vector representation approach, Mol2Vec. From a statistical perspective, MLP and GCN were found to perform superior over Mol2Vec, when applied to external validation sets. Interestingly, GCN-based predictions are most stable over a longer period in a time series validation study. Apart from those statistical observations, DNN prove of value to guide local SAR. To illustrate this important aspect in pharmaceutical research projects, we discuss challenging applications in medicinal chemistry towards a more realistic picture of artificial intelligence in drug discovery.

Trauma is a major risk factor for the development of a venous thromboembolism (VTE). After observing higher than expected VTE rates within our center's Trauma Quality Improvement Program data, we instituted a change in our VTE prophylaxis protocol, moving to enoxaparin dosing titrated by anti-Xa levels. We hypothesized that this intervention would lower our symptomatic VTE rates.Adult trauma patients at a single institution meeting National Trauma Data Standard criteria from April 2015 to September 2019 were examined with regards to VTE chemoprophylaxis regimen and VTE incidence. Two groups of patients were identified based on VTE protocol-those who received enoxaparin 30 mg twice daily without routine anti-Xa levels ("pre") versus those who received enoxaparin 40 mg twice daily with dose titrated by serial anti-Xa levels ("post"). Univariate and multivariate analyses were performed to define statistically significant differences in VTE incidence between the two cohorts.There were 1698 patients within the "pre" group and 1406 patients within the "post" group. The two groups were essentially the same in terms of demographics and risk factors for bleeding or thrombosis. There was a statistically significant reduction in VTE rate (p = 0.01) and deep vein thrombosis rate (p = 0.01) but no significant reduction in pulmonary embolism rate (p = 0.21) after implementation of the anti-Xa titration protocol. Risk-adjusted Trauma Quality Improvement Program data showed an improvement in rate of symptomatic pulmonary embolism from fifth decile to first decile.A protocol titrating prophylactic enoxaparin dose based on anti-Xa levels reduced VTE rates. Implementation of this type of protocol requires diligence from the physician and pharmacist team. Further research will investigate the impact of protocol compliance and time to appropriate anti-Xa level on incidence of VTE.Therapeutic/care management, Level IV.

Background and ObjectivesAndexanet alfa was recently approved as a reversal agent for the factor Xa inhibitors (FXais) apixaban and rivaroxaban, but its impact on long-term outcomes in FXai-associated intracerebral hemorrhage (ICH) is unknown. We aimed to explore potential clinical implications of andexanet alfa in FXai-associated ICH in this simulation study.MethodsWe simulated potential downstream implications of andexanet alfa across a range of possible hemostatic effects using data from a single center that treats FXai-associated ICH with prothrombin complex concentrate (PCC). We determined baseline probabilities of inadequate hemostasis across patients taking FXai and those not taking FXai via multivariable regression models and then determined the probabilities of unfavorable 3-month outcome (modified Rankin Scale score 4–6) using models comprising established predictors and each patient's calculated probability of inadequate hemostasis. We applied bootstrapping with model parameters from this derivation cohort to simulate a range of hemostatic improvements and corresponding outcomes and then calculated absolute risk reduction (relative to PCC) and projected number needed to treat (NNT) to prevent 1 unfavorable outcome.ResultsTraining models using real-world patients (n = 603 total, 55 on FXai) had good accuracy in predicting inadequate hemostasis (area under the curve [AUC] 0.78) and unfavorable outcome (AUC 0.78). Inadequate hemostasis was strongly associated with unfavorable outcome (odds ratio 4.5, 95% confidence interval [CI] 2.0–9.9) and occurred in 11.4% of patients taking FXai. Across simulated patients taking FXai comparable to those in A Study in Participants With Andexanet Alfa, a Novel Antidote to the Anticoagulation Effects of Factor Xa Inhibitors (ANNEXA-4) study, predicted absolute risk reduction of unfavorable outcome was 4.9% (95% CI 1.3%–7.8%) when the probability of inadequate hemostasis was reduced by 33% and 7.4% (95% CI 2.0%–11.9%) at 50% reduction, translating to projected NNT of 21 (cumulative cost $519,750) and 14 ($346,500), respectively.DiscussionEven optimistic simulated hemostatic effects suggest that the costs and potential benefits of andexanet alfa should be carefully considered. Placebo-controlled randomized trials are needed before its use can definitively be recommended.

Purpose Combination of Rivaroxaban plus Aspirin improved cardiovascular outcome in patients with stable cardiovascular disease. The aim was to determine if Rivaroxaban and acetylsalicylic acid alone or in combination may protect mitochondrial mitophagy in human coronary artery endothelial cells (HCAEC) exposed to D-glucose. Methods HCAEC were incubated under different conditions: 5 mmol/L glucose D-glucose (control), 30 mmol/L D-Glucose with and without 50 nmol/L Rivaroxaban (Rivaroxaban), 0.33 mmol/L ASA (ASA) or Rivaroxaban (12.5 nmol/L)+ASA (0.33 mmol/L; (Riva+ASA). Results HCAEC incubated with D-glucose showed an increased Factor Xa expression. The mitochondrial content of Pink-1 and Parkin were significantly reduced in high glucose-incubated HCAEC compared to control. Rivaroxaban+ASA significantly increased the mitochondrial content of Pink-1 and Parkin, and the mitochondrial membrane potential compared to D-Glucose group. Both ASA alone and Riva+ASA reduced reactive oxygen species (ROS) and tissue factor production induced by high glucose exposure. Conclusion Under high glucose condition combining Rivaroxaban+ASA increased the mitochondrial content of Pink-1 and Parkin, restored mitochondria membrane potential and reduced ROS and tissue factor expression in HCAEC. It suggests potential effects induced by dual use of Rivaroxaban and ASA on the coronary endothelium subjected to high glucose condition.

An accurate point-of-care test for detecting effective anticoagulation by direct oral anticoagulants (DOACs) in emergencies is an unmet need. We investigated the accuracy of a urinary qualitative strip test (DOAC Dipstick) to detect relevant DOAC exposure in patients who presented to an emergency department. In this prospective single-center cohort-type cross-sectional study, adults on DOAC treatment were enrolled. We assessed clinical sensitivity and specificity of DOAC Dipstick factor Xa and thrombin inhibitor pads to detect DOAC plasma levels ≥30 ng/mL using urine samples as the testing matrix. Liquid chromatography coupled with tandem-mass spectrometry was used as the reference standard method for plasma and urine measurement of DOAC concentrations. Of 293 patients enrolled, 265 patients were included in the analysis, of whom 92 were treated with rivaroxaban, 65 with apixaban, 77 with edoxaban, and 31 with dabigatran. The clinical sensitivity and specificity of the dipstick on urine samples to detect ≥30 ng/mL dabigatran plasma levels were 100% (95% confidence interval [CI]: 87–100%) and 98% (95% CI: 95–99%), respectively. The sensitivity and specificity of the dipstick to detect ≥30 ng/mL factor Xa inhibitor plasma levels were 97% (95% CI: 94–99%) and 69% (95% CI: 56–79%), respectively. The DOAC Dipstick sensitively identified effective thrombin and factor Xa inhibition in a real-world cohort of patients presenting at an emergency department. Therefore, the dipstick might provide a valuable test to detect relevant DOAC exposure in emergencies, although further studies will be needed to confirm these findings.

Current guidelines recommend monitoring the anticoagulant effect of unfractionated heparin (UFH) by measuring anti-Xa activity rather than activated partial thromboplastin time (aPTT) in intensive care unit (ICU) patients. The primary objective of this study was to evaluate the correlation of aPTT, anti-Xa activity, and thrombin generation in UFH-treated ICU patients. A prospective observational pilot study was conducted in adult surgical ICU patients treated with UFH. aPTT and anti-Xa activity were monitored daily. The therapeutic target was aPTT between 50 s and 84 s, and/or anti-Xa between 0.3 and 0.7 U/mL. Correlation among aPTT, anti-Xa activity, and thrombin generation was determined by measuring endogenous thrombin potential (ETP), with the inflammatory response evaluated. C-reactive protein (CRP) was used as a marker of inflammatory response. The plasma of 107 samples from 30 ICU patients was analyzed. The correlation between aPTT and anti-Xa activity was 0.66, CI95% [0.54;0.76] (p < 0.0001). Although thrombin generation, aPTT, and anti-Xa were correlated with inflammatory responses, the correlation was higher with thrombin generation and anti-Xa activity compared to aPTT. When aPTT was in a therapeutic range, a low thrombin generation was observed but was 50% inhibited when anti-Xa was in a therapeutic range. Coagulation testing with aPTT, anti-Xa correlated with thrombin generation. A 50% decrease in thrombin generation was observed when anti-Xa was within a therapeutic range. Further work is needed to evaluate coagulation biomarker responses and clinical outcomes in specific ICU populations.

There has been a landmark shift in the last several decades in the management and prevention of thromboembolic events. From the discovery of parenteral and oral agents requiring frequent monitoring as early as 1914, to the development of direct oral anticoagulants (DOACs) that do not require monitoring or dose adjustment in the late 20th century, great advances have been achieved. Despite the advent of these newer agents, bleeding continues to be a key complication, affecting 2 to 4% of DOAC-treated patients per year. Bleeding is associated with substantial morbidity and mortality. Although specific reversal agents for DOACs have lagged the release of these agents, idarucizumab and andexanet alfa are now available as antagonists. However, the efficacy of these reversal agents is uncertain, and complications, including thrombosis, have not been adequately explored. As such, guidelines continue to advise the use of nonspecific prohemostatic agents for patients requiring reversal of the anticoagulant effect of these drugs. As the indications for DOACs and the overall prevalence of their use expand, there is an unmet need for further studies to determine the efficacy of specific compared with nonspecific pro-hemostatic reversal agents. In this review, we will discuss the evidence behind specific and nonspecific reversal agents for both parenteral and oral anticoagulants.

Direct oral anticoagulants (DOACs) are increasingly used for stroke prevention in atrial fibrillation and treatment and prevention of venous thromboembolism. They are also associated with bleeding risk. Existing literature suggests that prothrombin complex concentrate (PCC) administration may help control bleeding due to factor Xa inhibitors (FXaI). To determine the hemostatic efficacy of PCC in patients with major bleeding due to FXaI, we performed a retrospective chart review of 50 patients who presented with FXaI associated major bleeding that required urgent hemostatic management. Hemostatic assessment was performed using ISTH and ANNEXA-4 criteria. Twenty patients presented with intracranial hemorrhage (ICH), 20 had gastrointestinal bleeding, 3 had visceral bleeding, 3 had genitourinary bleeding, and 4 had miscellaneous types of bleeding. Fifty-six percent (28/50) had effective hemostasis using ISTH criteria and 84% (42/50) achieved effective hemostasis by ANNEXA-4 criteria. Hemostatic efficacy was similar by both tools for ICH (75% each). However, there was a major difference between ISTH and ANNEXA-4 hemostatic efficacy assessments for GI bleeding (45% and 95%, respectively). When comparing rivaroxaban and apixaban, there was no significant difference in effective hemostasis using either criteria, time to hemostasis, thromboembolic events, or patient mortality. Five (10%) patients had thromboembolic events within seven days of PCC administration, and the 30-day mortality rate was 14% (7/50). Our study shows similar efficacy, thromboembolic events, and mortality associated with PCC compared to andexanet alfa using ANNEXA-4 criteria, suggesting that PCC may be a viable alternative.

The alterations in the hemostatic balance in COVID‐19 patients are strongly disturbed and contribute to a high prothrombotic status. The high rate of venous thromboembolism in COVID‐19 patients goes along with derangements in coagulation laboratory parameters. Hemostasis testing has an important role in diagnosed COVID‐19 patients. Elevated D‐dimer levels were found to be a crucial laboratory marker in the risk assessment of thrombosis in COVID‐19 patients. The diagnostic approach also includes prothrombin time and platelet count. Fibrinogen might give an indication for worsening coagulopathy. Other markers (activated partial thromboplastin time (aPTT), fibrinolysis parameters, coagulation factors, natural anticoagulants, antiphospholipid antibodies and parameters obtained by thromboelastography or thrombin generation assays) have been described as being deranged. These may help to understand the pathophysiology of thrombosis in COVID‐19 patients but have currently no place in diagnosis or management in COVID‐19 patients. For monitoring the heparin anticoagulant therapy, the anti‐Xa assay is suggested, because the severe acute‐phase reaction (high fibrinogen and high factor VIII) shortens the aPTT.

Background Direct oral anticoagulants (DOACs) have shown a positive benefit-risk balance in both clinical trials and real-world data, but approximately 2% to 3.5% of patients experience major bleeding annually. Many of these patients require hospitalization, and the administration of reversal agents may be required to control bleeding. Objectives The aim of this study was to investigate clinical outcomes associated with the use of 4-factor prothrombin complex concentrates, idarucizumab, or andexanet for reversal of severe DOAC-associated bleeding. Methods The investigators systematically searched for studies of reversal agents for the treatment of severe bleeding associated with DOAC. Mortality rates, thromboembolic events, and hemostatic efficacy were meta-analyzed using a random effects model. Results The investigators evaluated 60 studies in 4,735 patients with severe DOAC-related bleeding who were treated with 4-factor prothrombin complex concentrates (n = 2,688), idarucizumab (n = 1,111), or andexanet (n = 936). The mortality rate was 17.7% (95% confidence interval [CI]: 15.1% to 20.4%), and it was higher in patients with intracranial bleedings (20.2%) than in patients with extracranial hemorrhages (15.4%). The thromboembolism rate was 4.6% (95% CI: 3.3% to 6.0%), being particularly high with andexanet (10.7%; 95% CI: 6.5% to 15.7%). The effective hemostasis rate was 78.5% (95% CI: 75.1% to 81.8%) and was similar regardless of the reversal agent considered. The rebleeding rate was 13.2% (95% CI: 5.5% to 23.1%) and 78% of rebleeds occurred after resumption of anticoagulation. The risk of death was markedly and significantly associated with failure to achieve effective hemostasis (relative risk: 3.63; 95% CI: 2.56 to 5.16). The results were robust regardless of the type of study or the hemostatic scale used. Conclusions The risk of death after severe DOAC-related bleeding remains significant despite a high rate of effective hemostasis with reversal agents. Failure to achieve effective hemostasis strongly correlated with a fatal outcome. Thromboembolism rates are particularly high with andexanet. Comparative clinical trials are needed.

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

Background. Mitochondria have been involved in host defense upon viral infections. Factor Xa (FXa), a coagulating factor, may also have influence on mitochondrial functionalities. The aim was to analyze if in human pulmonary microvascular endothelial cells (HPMEC), the SARS-CoV-2 (COVID-19) spike protein subunits, S1 and S2 (S1+S2), could alter mitochondrial metabolism and what is the role of FXA. Methods. HPMEC were incubated with and without recombinants S1+S2 (10 nmol/L each). Results. In control conditions, S1+S2 failed to modify FXa expression. However, in LPS (1 μg/mL)-incubated HPMEC, S1+S2 significantly increased FXa production. LPS tended to reduce mitochondrial membrane potential with respect to control, but in higher and significant degree, it was reduced when S1+S2 were present. LPS did not significantly modify cytochrome c oxidase activity as compared with control. Addition of S1+S2 spike subunits to LPS-incubated HPMEC significantly increased cytochrome c oxidase activity with respect to control. Lactate dehydrogenase activity was also increased by S1+S2 with respect to control and LPS alone. Protein expression level of uncoupled protein-2 (UCP-2) was markedly expressed when S1+S2 were added together to LPS. Rivaroxaban (50 nmol/L), a specific FXa inhibitor, significantly reduced all the above-mentioned alterations induced by S1+S2 including UCP-2 expression. Conclusions. In HPMEC undergoing to preinflammatory condition, COVID-19 S1+S2 spike subunits promoted alterations in mitochondria metabolism suggesting a shift from aerobic towards anaerobic metabolism that was accompanied of high FXa production. Rivaroxaban prevented all the mitochondrial metabolic changes mediated by the present COVID-19 S1 and S2 spike subunits suggesting the involvement of endogenous FXa.

Background The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation.Objective We aimed to unravel the structure-function relationships of TIX-5.Method We used a structure model generated based on homology with the allergen Der F7.Results Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system.Conclusion The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.

Oral anticoagulant therapy has changed by clinical evidence that coagulation factor Xa (FXa) can be safely and effectively targeted for thromboprophylaxis. Because thrombotic and thrombo-inflammatory diseases are frequently caused by excessive activation of the tissue factor (TF) pathway, activation of FX by the TF-FVIIa complex is of central importance for understanding the roles of FXa in thrombosis and hemostasis and functions beyond blood coagulation. Recent data showed that the nascent product FXa associated with TF-FVIIa not only supports hemostatic cofactor VIII activation, but also broadly influences immune reactions in inflammation, cancer, and autoimmunity. These signaling functions of FXa are mediated through protease activated receptor (PAR) cleavage and PAR2 activation occurs in extravascular environments specifically by macrophage synthesized FX. Cell autonomous FXa-PAR2 signaling is a mechanism for tumor-promoting macrophage polarization in the tumor microenvironment and tissue penetrance of oral FXa inhibitors favors the reprogramming of tumor-associated macrophages for non-coagulant therapeutic benefit. It is necessary to decipher the distinct functions of coagulation factors synthesized by the liver for circulation in blood versus those synthesized by extrahepatic immune cells for activity in tissue milieus. This research will lead to a better understanding of the broader roles of FXa as a central regulator of immune and hematopoietic systems.

Blood coagulation factor X/Xa sits at a pivotal point in the coagulation cascade and has a role in each of the three major pathways (intrinsic, extrinsic and the common pathway). Due to this central position, it is an attractive therapeutic target to either enhance or dampen thrombin generation. In this brief review, I will summarize key developments in the molecular understanding of this critical clotting factor and discuss the molecular basis of FX deficiency, highlight difficulties in expressing recombinant factor X, and detail two factor X variants evaluated clinically.

Extracorporeal membrane oxygenation (ECMO) support has a high incidence of both bleeding and thrombotic complications. Despite clear differences in patient characteristics and pathologies between veno-venous (VV) and veno-arterial (VA) ECMO support, anticoagulation practices are often the same across modalities. Moreover, there is very little data on their respective coagulation profiles and comparisons of thrombin generation in these patients. This study compares the coagulation profile and thrombin generation between patients supported with either VV and VA ECMO. A prospective cohort study of patients undergoing VA and VV ECMO at an Intensive care department of a university hospital and ECMO referral centre. In addition to routine coagulation testing and heparin monitoring per unit protocol, thromboelastography (TEG), multiplate aggregometry (MEA), calibrated automated thrombinography (CAT) and von-Willebrand’s activity (antigen and activity ratio) were sampled second-daily for 1 week, then weekly thereafter. VA patients had significantly lower platelets counts, fibrinogen, anti-thrombin and clot strength with higher d-dimer levels than VV patients, consistent with a more pronounced consumptive coagulopathy. Thrombin generation was higher in VA than VV patients, and the heparin dose required to suppress thrombin generation was lower in VA patients. There were no significant differences in total bleeding or thrombotic event rates between VV and VA patients when adjusted for days on extracorporeal support. VA patients received a lower median daily heparin dose 8500 IU [IQR 2500–24000] versus VV 28,800 IU [IQR 17,300–40,800.00]

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial–mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)–mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216–GSK-3β, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-β– and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae–mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

Background and Purpose: Andexanet was approved by the Food and Drug Administration in 2018 for reversal of life-threatening or uncontrolled bleeding associated with factor Xa anticoagulation; however, the cost-effectiveness of Andexanet compared with standard of care (ie, prothrombin complex concentrate, PCC) in patients with factor Xa–associated intracranial hemorrhage (ICrH) is unknown. Methods: Cost-effectiveness analysis using a Markov cohort decision analytic model with a lifetime horizon was completed to determine the costs and benefits of Andexanet compared with PCC for reversal of factor Xa–associated ICrH. The population of interest was patients living in Canada on chronic factor Xa inhibitors for prevention of ischemic stroke in nonvalvular atrial fibrillation or the prevention/treatment of venous thromboembolism, presenting with an ICrH. Outcomes of interest were life expectancy (measured in years), quality-adjusted life years (QALY), costs (reported in 2020 Canadian dollars), and the incremental cost-effectiveness ratio. Results: An overall reduction in fatal ICrH and increase in thromboembolic events was associated with Andexanet compared with PCC. Andexanet had the highest discounted life expectancy of 2.53 years and a discounted QALY of 1.55. PCC had a discounted life expectancy of 2.09 years and a discounted QALY of 1.28. The average discounted lifetime costs were $237 177 Canadian dollars for Andexanet and $177 871 Canadian dollars for PCC. The strategy of Andexanet had an incremental cost-effectiveness ratio was $219 652 per QALY gained compared with the comparator of PCC. The probabilistic sensitivity analyses demonstrated that Andexanet (at its current cost) was cost-effective in 19% of simulations using a willingness-to-pay threshold of $50 000/QALY and 33% of simulations at $150 000/QALY. A 1-way sensitivity analysis found that for the incremental cost-effectiveness ratio to be Conclusions: Based on available evidence, Andexanet represents low value for reversal of factor Xa–associated ICrH; however, there is substantial uncertainty reflecting the currently available data. Further comparative evidence and costing data will become available in the future with randomized trials of Andexanet versus PCC.

The objective of this study was to analyze whether compression stocking therapy in the human varicose vein wall may change the levels of biomarkers associated with vein insufficiency.Dilated collateral varicose vein samples were obtained from patients showing chronic venous disease (class 2 of the Clinical, Etiology, Anatomy, and Pathophysiology classification). Before elective surgery, 12 patients underwent compression stocking therapy (for 1 month) and 9 patients did not (control group). Expression levels of biomarkers associated with endothelial functionality (nitric oxide synthase 3), inflammation (interleukin-6, interleukin-10), oxidative stress (Gp91Compression stockings increased the content of nitric oxide synthase 3 (control, 16.48 [16.04-17.40] AU; compression, 83.71 [67.70-91.85] AU; P .001) in the varicose vein wall that was accompanied by reduction of both interleukin-6 levels (control, 38.72 [33.48-48.52] pg/μg protein; compression, 14.49 [11.05-17.41] pg/μg protein; P = .001) and the expression of Gp91In the varicose vein wall, compression stocking therapy improved the content levels of biomarkers associated with endothelial functionality, inflammation, oxidative stress, and coagulation.

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.

Objective To explore the mode of action (MoA) by which sugammadex interferes with coagulation. Materials and methods 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. Results 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). Conclusion 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.

BACKGROUND: Heparin enhances the ability of the plasma protease inhibitor, antithrombin, to neutralize coagulation factor Xa and thrombin. Skeletal muscle myosin binds unfractionated heparin. OBJECTIVES: The aim of this study was to investigate the influence of myosin binding to heparin on antithrombin’s anticoagulant activity. METHODS: Inhibition of factor Xa and thrombin by antithrombin in the presence of different heparins and skeletal muscle myosin or cardiac myosin was studied by measuring inhibition of each enzyme’s chromogenic substrate hydrolysis. RESULTS AND CONCLUSIONS: Skeletal muscle myosin and cardiac myosin neutralized unfractionated heparin’s enhancement of antithrombin’s inhibition of purified factor Xa and thrombin. Skeletal muscle myosin also reduced the inhibition of factor Xa and thrombin by antithrombin in the presence of heparan sulfate. These two myosins did not protect factor Xa from antithrombin inhibition when tested in the presence of smaller heparins, e.g., low molecular weight heparin or heparin pentasaccharide. This chain length dependence for skeletal muscle myosin’s ability to reduce heparin’s anticoagulant activity might have potential implications for therapy for patients who experience increases in plasma myosin levels, e.g., acute trauma patients. In addition to the chain length, the type and extent of sulfation of glycosaminoglycans influenced the ability of skeletal muscle myosin to neutralize the polysaccharide’s ability to enhance antithrombin’s activity. In summary, these studies show that skeletal muscle myosin and cardiac myosin can influence antithrombin’s anticoagulant activity against factor Xa and thrombin, implying that they may significantly influence the hemostatic balance involving bleeding versus clotting.

Thrombin generation is pivotal to both physiological blood clot formation and pathological development of disseminated intravascular coagulation (DIC). In critical illness, extensive cell damage can release histones into the circulation, which can increase thrombin generation and cause DIC, but the molecular mechanism is not clear. Typically, thrombin is generated by the prothrombinase complex, comprising activated factor X (FXa), activated cofactor V (FVa), and phospholipids to cleave prothrombin in the presence of calcium. In this study, we found that in the presence of extracellular histones, an alternative prothrombinase could form without FVa and phospholipids. Histones directly bind to prothrombin fragment 1 (F1) and fragment 2 (F2) specifically to facilitate FXa cleavage of prothrombin to release active thrombin, unlike FVa, which requires phospholipid surfaces to anchor the classical prothrombinase complex. In vivo, histone infusion into mice induced DIC, which was significantly abrogated when prothrombin F1 + F2 were infused prior to histones, to act as decoy. In a cohort of intensive care unit patients with sepsis (n = 144), circulating histone levels were significantly elevated in patients with DIC. These data suggest that histone-induced alternative prothrombinase without phospholipid anchorage may disseminate intravascular coagulation and reveal a new molecular mechanism of thrombin generation and DIC development. In addition, histones significantly reduced the requirement for FXa in the coagulation cascade to enable clot formation in factor VIII (FVIII)– and FIX-deficient plasma, as well as in FVIII-deficient mice. In summary, this study highlights a novel mechanism in coagulation with therapeutic potential in both targeting systemic coagulation activation and correcting coagulation factor deficiency.

BACKGROUND/OBJECTIVE: There are limited data on the risks and benefits of using andexanet alfa (AA) in comparison to four-factor prothrombin complex concentrate (4F-PCC) to reverse Factor Xa inhibitors (FXi) associated intracranial hemorrhage (ICH). We sought to describe our experience with AA or 4F-PCC in patients with oral FXi-related traumatic and spontaneous ICH. METHODS: We conducted a retrospective review of consecutive adult patients with FXi-related ICH who received AA or 4F-PCC. FXi-related ICH cases included traumatic and spontaneous intracranial hemorrhages. Our primary analysis evaluated ICH stability on head computed tomography scan (CT), defined as a similar amount of blood from the initial scan at the onset of ICH to subsequent scans, at 6 hours and 24 hours post-administration of AA or 4F-PCC. For the subset of spontaneous intraparenchymal hemorrhages, volume was measured at 6 hours and 24 hours post reversal. In secondary analyses, we evaluated good functional outcome at discharge, defined as a Modified Rankin Score (mRS) of less than 3, and the occurrence of thrombotic events after administration of AA or 4F-PCC during hospitalization. RESULTS: A total of 44 patients (16 traumatic and 28 spontaneous ICH) with median age of 79 years [72–86], 36% females, with a FXi-related ICH, were included in this study. The majority of spontaneous ICHs were intraparenchymal 19 (68%). Twenty-eight patients (64%) received AA and 16 patients (36%) received 4F-PCC. There was no difference between AA and 4F-PCC in terms of CT stability at 6 hours (21 [78%] vs 10 [71%], p = 0.71) and 24 hours (15 [88%] vs 6 [60%], p = 0.15). In a subgroup of patients with spontaneous intraparenchymal hemorrhage, there was no difference in the degree of achieved hemostasis based on hematoma volume between AA and 4F-PCC at 6 hours (9.3 mL [6.9–26.4] vs 10 mL [9.4–22.1], adjusted p = 0. 997) and 24 hours (9.2 mL [6.1–18.9] vs 9.9 [9.4–21.1], adjusted p =1). The number of patients with good outcome based on mRS on discharge were 10 (36%) and 6 (38%) in the AA and 4F-PCC groups, respectively (adjusted p = 0.81). The incidence of thromboembolic events was similar in the AA and 4F-PCC groups (2 [7%] vs 0, p = 0.53). CONCLUSION: In this limited sample of patients, we found no difference in neuroimaging stability, functional outcome, and thrombotic events when comparing AA and 4F-PCC in patients with FXi-related ICH. Since our analysis is likely underpowered, a multi-center collaborative network devoted to this question is warranted.

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.

Background There are inadequate data on the optimal strategy for transitioning factor Xa inhibitors (FXai; apixaban, rivaroxaban) to unfractionated heparin (UFH) infusions. Objective In patients transitioning from an FXai to an UFH infusion, this study compared the safety and efficacy of monitoring UFH infusions using an activated partial thromboplastin time (aPTT) titration scale versus utilizing an UFH-calibrated anti-Xa titration scale aided by a novel institutional guideline. Methods A retrospective cohort analysis was conducted on adult patients transitioning from an FXai to an UFH infusion at 2 medical centers from June 1, 2018, to November 1, 2020. One institution utilized aPTT while the other institution primarily used UFH-calibrated anti-Xa. The primary endpoint was a composite of death, major bleeding, or new thrombosis during the hospitalization with a planned noninferiority analysis. Secondary outcomes were also collected including the amount and duration of UFH administered between cohorts. Results The incidence rate of the primary composite endpoint was 6.3% in the anti-Xa group and 11% in the aPTT group ( P < 0.001 for noninferiority, P = 0.138 for superiority) meeting noninferiority criteria. No statistical differences were seen in new thrombosis, major bleeding, or any bleeding. Conclusion and Relevance This represents the first report of a comparison between aPTT versus anti-Xa monitoring in relation to clinical outcomes for patients transitioning from an FXai to an UFH infusion. A transition guideline primarily utilizing an UFH-calibrated anti-Xa assay appears to be a safe alternative to aPTT monitoring and can aid facilities in the management of patients during these complex transitions.

Background With a growing number of patients on new oral anticoagulants, interest in reversal agents is rising. Andexanet alfa is used for reversal of factor Xa inhibitors in intracranial hemorrhage. Methods We provide a brief review on andexanet-alfa-associated heparin resistance and discuss potentially critical situations from different clinical perspectives. Results Case reports point out that andexanet alfa can cause unresponsiveness to heparin, leading to catastrophic events. As a result, regulatory bodies have issued warning notices to avoid heparinization parallel to the use of andexanet alfa. Conclusions Although well known to hematologists, the phenomenon is underrecognized among stroke clinicians. However, patients with intracranial hemorrhage frequently undergo endovascular or surgical interventions that require periprocedural administration of heparin.

Andexanet alfa is approved (FDA "accelerated approval"; EMA "conditional approval") as the first specific reversal agent for factor Xa (FXa) inhibitor-associated uncontrolled or life-threatening bleeding. Four-factor prothrombin complex concentrates (4F-PCC) are commonly used as an off-label, non-specific, factor replacement approach to manage FXa inhibitor-associated life-threatening bleeding. We evaluated the effectiveness and safety of andexanet alfa versus 4F-PCC for management of apixaban- or rivaroxaban-associated intracranial hemorrhage (ICH).This two-cohort comparison study included andexanet alfa patients enrolled at US hospitals from 4/2015 to 3/2020 in the prospective, single-arm ANNEXA-4 study and a synthetic control arm of 4F-PCC patients admitted within a US healthcare system from 12/2016 to 8/2020. Adults with radiographically confirmed ICH who took their last dose of apixaban or rivaroxaban 24 h prior to the bleed were included. Patients with a Glasgow Coma Scale (GCS) score 7, hematoma volume 60 mL, or planned surgery within 12 h were excluded. Outcomes were hemostatic effectiveness from index to repeat scan, mortality within 30 days, and thrombotic events within five days. Odds ratios (ORs) with 95% confidence intervals (CI) were calculated using propensity score-overlap weighted logistic regression.The study included 107 andexanet alfa (96.6% low dose) and 95 4F-PCC patients (79.3% receiving a 25 unit/kg dose). After propensity score-overlap weighting, mean age was 79 years, GCS was 14, time from initial scan to reversal initiation was 2.3 h, and time from reversal to repeat scan was 12.2 h in both arms. Atrial fibrillation was present in 86% of patients. Most ICHs were single compartment (78%), trauma-related (61%), and involved the intracerebral and/or intraventricular space(s) (53%). ICH size was ≥ 10 mL in volume (intracerebral and/or ventricular) or ≥ 10 mm in thickness (subdural or subarachnoid) in 22% of patients and infratentorial in 15%. Andexanet alfa was associated with greater odds of achieving hemostatic effectiveness (85.8% vs. 68.1%; OR 2.73; 95% CI 1.16-6.42) and decreased odds of mortality (7.9% vs. 19.6%; OR 0.36; 95% CI 0.13-0.98) versus 4F-PCC. Two thrombotic events occurred with andexanet alfa and none with 4F-PCC.In this indirect comparison of patients with an apixaban- or rivaroxaban-associated ICH, andexanet alfa was associated with better hemostatic effectiveness and improved survival compared to 4F-PCC. Trial registration NCT02329327; registration date: December 31, 2014.

The article is an opinion on the problem of venous thromboembolic (VTE) complications in patients with chronic kidney disease (CKD), which is significant and urgent for Russia. Signs of CKD are noted in more than 1/3 of patients with chronic heart failure; a decrease in kidney function is observed in 36% of people over the age of 60, in people of working age, a decrease in function is noted in 16% of cases, and in the presence of cardiovascular diseases increases to 26%. Clinical research data convincingly show that CKD is an independent risk factor for the development of VTE complications. The last decade has given us the opportunity to observe a kind of "revolution" in VTE therapy, which is associated with the appearance on the market of direct oral anticoagulants, including inhibitors of factor IIa (thrombin) and factor Xa. These drugs are approved by the Food and Drug Administration for the treatment of acute thromboembolism. Nevertheless, patients with severe CKD (estimated glomerular filtration rate 30 ml/min) are still limited to the use of unfractionated heparin and vitamin K antagonists, as there is insufficient data to support the use of direct oral anticoagulants in this population.Статья представляет собой обсуждение проблемы венозных тромбоэмболических осложнений (ВТЭО) у больных хронической болезнью почек (ХБП), которая для нашей страны является значимой и насущной. Признаки ХБП отмечаются более чем у 1/3 пациентов с хронической сердечной недостаточностью; снижение функции почек наблюдается у 36% лиц в возрасте старше 60 лет, у лиц трудоспособного возраста снижение функции отмечается в 16% случаев, а при наличии сердечно-сосудистых заболеваний возрастает до 26%. Данные клинических исследований убедительно показывают, что ХБП является независимым фактором риска развития ВТЭО. Последнее десятилетие дало нам возможность наблюдать некую революцию в терапии ВТЭО, которая связана с появлением на рынке прямых пероральных антикоагулянтов, включающих ингибиторы фактора IIa (тромбина) и фактора Ха. Эти препараты одобрены Управлением по контролю пищевых продуктов и лекарств в США для лечения острой тромбоэмболии. Тем не менее пациенты с тяжелой ХБП (расчетная скорость клубочковой фильтрации 30 мл/мин) по-прежнему ограничены использованием нефракционированного гепарина и антагонистов витамина К, так как недостаточно данных в поддержку использования прямых пероральных антикоагулянтов в этой популяции.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.