Anti-factor Xa Activity Research Articles

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Heparin resistance in severe thermal injury: A prospective cohort study.

BackgroundLow molecular-weight heparin (LMWH) is routinely administered to burn patients for thromboprophylaxis. Some studies have reported heparin resistance, yet the mechanism(s) and prevalence have not been systematically studied. We hypothesized that nucleosomes, composed of histone structures with associated DNA released from injured tissue and activated immune cells in the form of neutrophil extracellular traps (NETs or NETosis), neutralize LMWH resulting in suboptimal anticoagulation, assessed by reduction in anti-factor Xa activity.MethodsBlood was sampled from >15% total body surface area (TBSA) burn patients receiving LMWH on days 5, 10 and 14. Peak anti-factor Xa (AFXa) activity, anti-thrombin (ATIII) activity, cell-free DNA (cfDNA) levels and nucleosome levels were measured. Mixed effects regression was adjusted for multiple confounders, including injury severity and ATIII activity, and was used to test the association between nucleosomes and AFXa.ResultsA total of 30 patients with severe burns were included. Mean TBSA 43% (SD 17). Twenty-three (77%) patients were affected by heparin resistance (defined by AFXa activity <0.2 IU/mL). Mean peak AFXa activity across samples was 0.18 IU/mL (SD 0.11). Mean ATIII was 81.9% activity (SD 20.4). Samples taken at higher LWMH doses were found to have significantly increased AFXa activity, though the effect was not observed at all doses, at 8000 IU no samples were heparin resistant. Nucleosome levels were negatively correlated with AFXa (r = −0.29, p = 0.050) consistent with the hypothesis. The final model, with peak AFXa as the response variable, was adjusted for nucleosome levels (p = 0.0453), ATIII activity (p = 0.0053), LMWH dose pre-sample (p = 0.0049), drug given (enoxaparin or tinzaparin) (p = 0.03), and other confounders including severity of injury, age, gender, time point of sample.ConclusionsHeparin resistance is a prevalent issue in severe burns. Nucleosome levels were increased post-burn, and showed an inverse association with AFXa consistent with the hypothesis that they may interfere with the anticoagulant effect of heparin in vivo and contribute to heparin resistance. Accurate monitoring of AFXa activity with appropriate therapy escalation plans are recommended with dose adjustment following severe burn injury.

Is specific anti-factor Xa activity measurement a way to predict relevant bleeding in non-valvular atrial fibrillation (NVAF) patients?

Anti-Xa direct oral anticoagulants (DOACs) are first line therapy for stroke prevention in NVAF patients. There is no need of regular anticoagulation monitoring and dose reduction may be indicated in patients showing a high bleeding risk. However, we still estimate 2 to 5% of major bleeding (MB) and clinically relevant non-major bleeding (CRNMB) in NVAF patients. Thereupon, we must identify other bleeding risks such as biological overdose. The goal of this study was to investigate specific anti-Xa activity measurement as a mean to predict bleeding complications in NVAF patients. One hundred and forty-six patients (either on apixaban or rivaroxaban) were enrolled from 2017 to 2019. The DOACs measurements were performed the day of inclusion. We determined an overdose and therapeutic underdose thresholds according to expected anti-Xa plasma range levels filled in the European heart rhythm association guide use of DOACs. MB, CRNMB and minor bleedings and strokes occurring during the follow up (up to 3 years) were recorded. We analysed the correlation between therapeutic overdose and the studied endpoints. To this day, the statistical analyses are still being conducted. The first results show that among the 146 patients, more or less 14% were considered overdosed. Moreover, a statistical trend was found between overdosed patients and MB or CRNMB. In routine clinical practice, anti-Xa activity measurement could be used to predict bleeding risk in a selected population in order to adapt doses or to consider a change of molecule.

Is specific anti-factor Xa activity measurement a way to predict relevant bleedings in non-valvular atrial fibrillation (NVAF) patients?

Direct oral anticoagulants (DOACs) are first line therapy for stroke prevention in NVAF patients. They were reported to be as effective as vitamin K antagonists (VKA) for stroke prevention in high-risk NVAF patients, with lower bleeding events. Compared to VKA, there is no need of regular anticoagulation monitoring. Yet, we still estimate 2 to 5% of major bleedings (MB) and up to 15% of clinically relevant non-major bleedings (CRNMB). The goal of this study was to investigate specific anti-Xa activity (AXA) measurement as a mean to predict bleeding complications in NVAF patients. We also determined anti-Xa overdose risk factors. We performed a prospective observational study from December 2016 to April 2020. We took blood samples on patients the day of inclusion to measure AXA. We considered as high responders, patients to whom AXA plasma levels were superior to the upper limit of the expected plasma level ranges from the European Heart Rhythm Association recommendations. MB, CRNMB from time to inclusion to April 2020 were collected. We then compared group of high responders and non-high responders. A total of 146 patients were analysed. Mean follow up was 20 months. Among them, 24 (16,4%) had MB or CRNMB during the follow up. 36 (24,6%) patients were high responders among which 10 had relevant bleedings. High responders showed a higher risk of bleeding in univariate analyses ( P = 0.034). After adjustment in multivariate analyses, only HASBLED had an increased risk of bleeding with an OR of 2.88 (1.74–5.12); probably due to a small number of patients and short time of follow up. We also showed that women and concomitant use of amiodarone were responsible for increasing AXA plasma level. AXA plasma level might be predictive of bleeding. Further studies with higher number of patients should be carried out to prove it. Female and amiodarone are both overdose risk factors.

Assessment of anti-factor Xa activity in critically ill COVID-19 patients receiving three different anticoagulation regimens

Introduction:Critically ill COVID-19 patients are at increased risk of thrombosis with an enhanced risk of bleeding. We aimed to explore the role of anti-factor Xa levels in optimizing the high-intensity anticoagulation’s safety and efficacy and finding possible associations between D-dimer levels, cytokine storm markers, and COVID-19-induced coagulopathy or thrombophilia.Methods:Retrospective cohort study conducted on 69 critically ill COVID-19 patients who received three regimens of higher intensity anticoagulation.Results:Seventeen patients (24.6%) received high-dose enoxaparin prophylaxis, 29 patients (42%) received therapeutic doses of enoxaparin, and 23 patients (33.3%) were on therapeutic unfractionated heparin infusion. Fewer than one-third of the whole cohort (n = 22; 31.8%) achieved the target range of anti-factor Xa. The patients were divided into three subgroups based on anti-factor Xa target status within each anticoagulation regimen; when compared, the only association observed among them was for interleukin-6 levels, which were significantly higher in both the “above the expected range” and “below the expected range” groups compared with the “within the expected range” group (p = 0.009). Major bleeding episodes occurred in 14 (20.3%) patients and were non-significantly more frequent in the “below the expected anti-factor Xa range group” (p = 0.415). Seven patients (10.1%) developed thrombosis. The majority of patients had anti-factor Xa levels below the expected ranges (four patients, 57.1%).Conclusion:Conventional anti-factor Xa ranges may not be appropriate as a predictive surrogate for bleeding in critically ill COVID-19. The clinical decision to initiate therapeutic anticoagulation preemptively may be individualized according to thrombosis and bleeding risks. Cytokine storm markers, namely, interleukin-6, may play a role in COVID-19-induced coagulopathy or thrombophilia.

Дозирование низкомолекулярных гепаринов и анти-фактор Xa активность у пациентов с новой коронавирусной инфекцией COVID-19.

Дозирование низкомолекулярных гепаринов и анти-фактор Xa активность у пациентов с новой коронавирусной инфекцией COVID-19.

Sulfated Derivatives of Arabinogalactan and Their Anticoagulant Activity

A comparison of IR spectra and molecular weight distribution of arabinogalactan sulfates as sodium and ammonium salts obtained using various sulfating reagents was performed. According to the data, sulfated derivatives of arabinogalactan differ from each other in the nature of hydrogen bonds and molecular weight distribution. Using coagulological tests during activation of coagulation of platelet-poor human plasma in vitro, the anticoagulant properties of sulfated arabinogalactan derivatives in various salt forms differing in the preparation method, sulfation degree, and molecular weight were studied. It was found that the sample in the form of the sodium salt of sulfated arabinogalactan (SAG 1) with a sulfur content of 13.2 wt % and a polydispersity degree of 1.52 exhibited twofold greater anticoagulant activity than the sample in the form of the ammonium salt of sulfated arabinogalactan (SAG 2) with a sulfur content of 6.6 wt % and a polydispersity degree of 1.30. The antithrombin (aIIa) activity of the samples obtained by sulfation with a complex of pyridine and sulfuric anhydride (SAG 1) and a complex of sulfamic acid (SAG 2) was 23.42 ± 1.86 and 10.20 ± 1.50 U/mg, respectively; the value of the antifactor Xa activity of SAG 1 and SAG 2 was 2.13 ± 0.42 and 0.37 ± 0.08 U/mg; the aIIa/aXa ratio for SAG 1 and SAG 2 was 11 and 28, respectively. The antifactor Xa (aXa) activity of SAG, which is lower than that of unfractionated heparin (UFH), and a high aIIa/aXa activity ratio may contribute to less provocation of bleeding by SAG samples compared to UFH.

606 Oral anticoagulants in fragile patients with percutaneous endoscopic gastrostomy and atrial fibrillation: the Origami study

Abstract Aims Randomized trials support the safety and efficacy of direct oral anticoagulants (DOACs) vs. vitamin K antagonists (VKA) in patients with nonvalvular atrial fibrillation (AF), leading to increased use of these compounds. Crushed forms of DOACs have shown to be reliable, but evidence supporting percutaneous endoscopic gastrostomy (PEG) delivery is lacking. PEG is a long-term option for enteral food and drug delivery in patients unable to maintain oral intake, bypassing the risks and disadvantages of parenteral nutrition. We investigate the safety and efficacy of Edoxaban administered via PEG in patients with atrial fibrillation and a clinical indication for a long-term anticoagulation. Methods and results In this prospective, single centre observational study, 12 PEG-treated patients with indication to anticoagulation will receive edoxaban via PEG and will be followed-up to 6 months. Plasma anti-Factor Xa activity and edoxaban concentrations will be assessed. Thromboembolic (ischaemic stroke, systemic embolism, venous thromboembolism) and bleeding events (Bleeding Academic Research Consortium and Thrombolysis in Myocardial Infarction) will be recorded at 1 and 6 months. A retrospective analysis of 5 AF cases undergoing PEG-implantation at our Institution, who received edoxaban via PEG, showed plasma anti-FXa levels at steady state of 146 ± 15 ng/ml, without major adverse event at a mean follow-up of 6 months. Conclusion We prospectively investigate PEG-administration of edoxaban in PEG-treated patients requiring long term anticoagulation. Our preliminary retrospective data support this route of DOAC administration.

Utility of coagulation analyses to assess thromboprophylaxis with dalteparin in intensive care unit patients.

The aim of this study was to evaluate the utility of coagulation analyses to assess thromboprophylaxis with dalteparin in intensive care unit (ICU) patients. Prospective observational study of ICU patients receiving dalteparin prophylaxis at Oslo University Hospital in Norway. Trough and peak antithrombin, protein C, anti-factor Xa activity (aFXa), d-dimer, thromboelastography, calibrated automated thrombogram and microparticles were analysed. Levels were compared in patients with and without venous thromboembolism (VTE), major bleeding, acute kidney injury (AKI) with use of renal replacement therapy (RRT) and variable dalteparin dose. Among 50 included patients (76% male, mean age 62years) five (10%) developed VTE and eight (16%) major bleeding. Median through aFXa level was 0.03 (0.02-0.05) IU/mL, and 48 (96%) of patients were within and two (4%) above target range. Peak aFXa level was 0.21 (0.13-0.29) IU/mL, the number of patients below, within and above prophylactic range were 21 (42%), 25 (50%) and four (8%). Peak aFXa levels were similar in patients with and without VTE (0.18 vs 0.21IU/L, P=.72), major bleeding (0.22 vs 0.21IU/mL, P=.38) and AKI with RRT (0.18 vs 0.24, P=.13), but lower in patients receiving dalteparin 5000IU od compared to 7500IU od (0.19 vs 0.30IU/mL, P<.01). Intensive care unit patients receiving dalteparin prophylaxis had half of patients within prophylactic peak aFXa target range. Peak aFXa levels was affected by administered dalteparin dose, but not presence of VTE, major bleeding or AKI with RRT.

Abstract 15194: The Distribution of Anti-factor Xa Activity Values, Prothrombin Time and Activated Partial Thromboplastin Time Assessed at Multiple Points in Patients on Edoxaban Therapy

Introduction: Edoxaban is one of the direct oral anticoagulants (DOACs) used for non-valvular atrial fibrillation (NVAF) or venous thromboembolism (VTE). Chromogenic anti-factor Xa activity (AXA) is the appropriate assay to measure the pharmacodynamics of a factor Xa inhibitor and to estimate plasma drug concentrations. Although patients taking DOACs do not require routine coagulation monitoring, the distributions of AXA values have not been assessed at multiple points in the previous study. Purpose: To clarify the distribution of AXA values at two-hour intervals in patients taking edoxaban. Methods: Sixteen patients (66.8 ± 9.6 years, 11 males) with NVAF or VTE on edoxaban therapy were enrolled in the study. We measured AXA, using chromogenic assay with the HemosIL Liquid Heparin kit, at two-hour intervals (from immediately before the intake of edoxaban to 8 hours after the intake). The dosage of edoxaban was 30 mg (n=11) once daily (OD) or 60 mg (n=5) OD according to the prescribing information. Results: The distributions of AXA values of 30 mg OD at 0, 2, 4, 6 and 8 hours after the intake of edoxaban were 0.12±0.08, 0.80±0.48, 0.94±0.48, 0.72±0.34 and 0.53±0.32 (IU/mL), respectively. The distributions of AXA values of 60 mg OD at 0, 2, 4, 6 and 8 hours after the intake of edoxaban were 0.17±0.04, 1.59±0.76, 1.43±0.34, 1.29±0.32 and 0.96±0.21 (IU/mL), respectively. Conclusions: Our findings reveal that the peak time of AXA values might be different depending on the dosage. The peak time of 30 mg OD is between 0 to 4 hours, whereas that of 60 mg OD is between 2 to 6 hours after the intake of edoxaban. In addition, the AXA values of 60 mg OD are almost twice as much as that of 30 mg at each point.

ORal anticoagulants In fraGile patients with percutAneous endoscopic gastrostoMy and atrIal fibrillation: the (ORIGAMI) study.

The ORal anticoagulants In fraGile patients with percutAneous endoscopic gastrostoMy and atrIal fibrillation (ORIGAMI) study investigates the safety and efficacy of Edoxaban administered via PEG in patients with atrial fibrillation and a clinical indication for a long-term anticoagulation. In this prospective, single-centre observational study, 12 PEG-treated patients with indication to anticoagulation will receive edoxaban via PEG and will be followed up to 6 months. Plasma antifactor Xa activity and edoxaban concentrations will be assessed. Thromboembolic (ischaemic stroke, systemic embolism, venous thromboembolism) and bleeding events (Bleeding Academic Research Consortium and Thrombolysis in Myocardial Infarction) will be recorded at 1 and 6 months. A retrospective analysis of five atrial fibrillation cases undergoing PEG implantation at our Institution who received edoxaban via PEG showed plasma anti-FXa levels at a steady state of 146 ± 15 ng/ml, without major adverse event at a mean follow-up of 6 months. ORIGAMI prospectively investigates PEG-administration of edoxaban in PEG-treated patients requiring long-term anticoagulation. Our preliminary retrospective data support this route of DOAC administration. NCT04271293.

The correlations between anti-factor Xa activity values and PT/APTT at peak and trough times in patients with venous thromboembolism using high dose of apixaban

Abstract Background The high dose (20mg/day) of apixaban is used for the initial treatment of venous thromboembolism for the first week. Although patients taking direct oral anticoagulants do not require routine coagulation monitoring, the correlations between anti-factor Xa activity (AXA) and routine coagulation markers such as prothrombin time (PT) and activated partial thromboplastin time (APTT) at peak and trough times especially when using high dose of apixaban have not been reported so far. Purpose The purpose is to assess the correlations between AXA values and PT/APTT at peak and trough times in patients with venous thromboembolism using high dose of Apixaban. Methods Twenty-six patients (10 male; 71±15 years) with proximal venous thromboembolism or pulmonary embolism using high dose (20mg/day) of apixaban were enrolled. We measured AXA, using chromogenic assay with the HemosIL Liquid Heparin kit, PT and APTT at peak and trough times. The peak time was defined as 3 hours after the intake of apixaban, and the trough time was defined as that immediately before the intake of apixaban. Results A significant and strong positive correlation was observed between AXA and PT at both peak and trough times (R=0.795, p&amp;lt;0.01 and R=0.766, p&amp;lt;0.01, respectively). A significant and moderate positive correlation was observed between AXA and APTT at trough time (R=0.527, p&amp;lt;0.01), but no correlation was observed between AXA and APTT at peak time (R=0.366, p=0.07). Conclusion Our findings reveal the relationship between AXA and PT at peak and trough times has a significant strong correlation. These results suggest measuring of PT may be alternative and effective way of monitoring of AXA values when using high dose of apixaban. Figure 1 Funding Acknowledgement Type of funding source: None

The distribution of anti-factor Xa activity value, PT and APTT at peak and trough times in patients with direct anti-factor Xa inhibitors

Abstract Background Although patients taking direct oral anticoagulants (DOACs) do not require routine coagulation monitoring, the distribution of anti-factor Xa activity (AXA) values, prothrombin time (PT), PT-international normalized ratio (INR) and activated partial thromboplastin time (APTT) in patients on apixaban, edoxaban and rivaroxaban therapy is still not clear. Purpose The aim is to set the standard values of AXA values, PT, PT-INR and APTT in patients using DOACs. Methods We measured AXA, using chromogenic assay with the HemosIL Liquid Heparin kit, PT, PT-INR and APTT at trough and peak times in 224 patients with non-valvular atrial fibrillation and venous thromboembolism, of whom 90 received apixaban, 100 received edoxaban and 34 received rivaroxaban. The peak time was defined as 3 hours after the intake of apixaban or rivaroxaban, and 2 hours after the intake of edoxaban. The trough time was defined as that immediately before the intake. The AXA values, PT, PT-INR and APTT were measured at least 72 hours after the start of treatment. The dosage of DOACs is defined according to the prescribing information in Japan. Results (The order of results below is apixaban, edoxaban and rivaroxaban, respectively.) The average AXA values were 2.29, 0.23 and 0.39 (IU/mL) at trough time, and 3.04, 1.01 and 1.70 (IU/mL) at peak time. The average PT values were 17.9, 12.9 and 13.1 (s) at trough time, and 19.7, 15.5 and 17.5 (s) at peak time. The average PT-INR values were 1.49, 1.07 and 1.08 at trough time, and 1.65, 1.29 and 1.45 at peak time. The average APPT values were 34.5, 31.3 and 32.0 (s) at trough time, and 39.5, 35.9 and 39.8 (s) at peak time. Conclusion Our findings reveal the standard values of AXA, PT, PT-INR and APTT in patients using apixaban, edoxaban and rivaroxaban in each dosage. The DOACs should be changed if the measured value is out of those standard values in 90% confidence interval. Funding Acknowledgement Type of funding source: None

The Influence of Haemostatic System Maturation on the Dose-Response Relationship of Unfractionated Heparin.

Unfractionated heparin (UFH) dosing and monitoring guidelines for children are often extrapolated from adult data. This practice is suboptimal given the inherent differences in haemostatic maturation and drug handling in children compared with adults. The aim of this work was to investigate the impact of haemostatic system maturation on the dose-response relationship of UFH in children. A quantitative model for haemostasis in adults was adapted to account for maturation in UFH pharmacokinetic (PK) parameters with and without age-related changes in coagulation factor concentrations. The adult and adapted models were used to predict the time courses of anti-factor Xa activity (aXa) and activated partial thromboplastin time (aPTT) in patients receiving UFH infusion. Predictions from both models were compared with observed aXa and aPTT measurements from 31 paediatric patients receiving UFH during extracorporeal membrane oxygenation (ECMO). The model with maturation for both UFH PK and the haemostatic system had an improved aXa and aPTT predictive performance compared with maturation in UFH PK only and the original adult model. Despite the minor effect of haemostatic system maturation on baseline aPTT, it led to substantial changes in the time course of aPTT sensitivity to UFH. This finding suggests that between-subject variability in clotting factors concentrations is potentially a major contributor to the overall variability of aPTT response to UFH. In addition, time-varying clotting factors concentrations may explain within-subject changes in aPTT sensitivity to UFH. We developed the first quantitative systems pharmacology (QSP) model that provides a mechanistic and quantitative basis for linking physiological and pharmacological maturation to UFH effect and response biomarkers. After appropriate clinical validation, the model could be useful for the development of paediatric-specific individualised UFH dosing recommendations.

Utilization and Laboratory Monitoring of Andexanet Alfa Reversal of Factor Xa Inhibitors in Bleeding Patients

Abstract Introduction Andexanet alfa, a reversal agent for factor Xa inhibitors rivaroxaban and apixaban, was approved by the Food and Drug Administration in May 2018 for patients with life-threatening bleeding. It acts by competing with endogenous factor Xa to block anticoagulant activity. Intravenous administration rapidly reduces anti-factor Xa activity. Because andexanet became available only recently, there is high potential for inappropriate use. Our objective was to investigate utilization and laboratory monitoring of andexanet in our hospital system in order to improve utilization. Methods This is a retrospective study with review of charts in the electronic medical record from April 2019 to February 2020. Charts were reviewed for the time of andexanet administration, anti-factor Xa testing before and after administration, turnaround time (TAT) for anti-factor Xa testing, and patient’s status after treatment. Results We identified 33 patients who received andexanet in our hospital system. Intracranial hemorrhage was the most common clinical symptom. Of 33 patients, 12 patients (36%) expired, one transferred to hospice, 18 (55%) were discharged alive, and two recent cases are alive awaiting disposition. Only 12 patients (36%) had anti-factor Xa testing performed prior to administration of andexanet, with one additional patient receiving testing only after administration. Factor Xa activity was misordered for anti-factor Xa in one patient. TAT for anti-factor Xa in these patients ranged from 25–112 minutes, with median 35.5 minutes. Of the 12 patients who had anti-factor Xa testing prior to administration, 4 expired and 8 survived. Pre-administration anti-factor Xa activity was significantly lower in patients who expired than in patients who survived (0.3 vs. 1.8 IU/ml, P value = 0.02). Conclusion Currently, anti-factor Xa testing is not ordered for most patients receiving andexanet alfa in our hospital system. Although not necessary for dosing decisions, anti-factor Xa testing may be useful prior to administration to identify patients unlikely to benefit from anticoagulant reversal. Despite a small sample size, there was a statistically significant difference in anti-factor Xa activity by patient outcome, suggesting that andexanet may be unhelpful in patients with low anti-factor Xa activity. Based on these findings, we are building an electronic order set to facilitate proper administration of this medication.

Elevated eosinophil count is related with lower anti-factor Xa activity in COVID-19 patients.

Despite prophylactic anticoagulant treatments, thrombotic complications may develop in patients with coronavirus disease 2019 (COVID-19). This study aimed to evaluate the factors influencing anti-factor Xa activity in COVID-19 patients receiving low molecular weight heparin (LMWH). We prospectively evaluated 80 COVID-19 patients, diagnosed using polymerase chain reaction test, who were admitted to our clinic and administered LMWH; LMWH (enoxaparin) was applied according to the weight, D-dimer levels, and clinical condition of patients. Anti-factor Xa activity in blood, drawn 4 h after the 3rd dose of LMWH, was measured and an activity of < 0.2 IU/mL was considered subprophylactic. Patients were followed up clinically, and anti-factor Xa activity was re-examined before discharge. Groups 1 and 2 included 13 and 67 patients with subprophylactic (mean ± SD: 0.18 ± 0.06) and prophylactic (mean ± SD: 0.43 ± 0.23) anti-factor Xa activity, respectively. The proportion of eosinophils in patients was significantly higher in group 1 than in group 2 (mean ± SD; 2.96 ± 2.55 vs 0.90 ± 1.28; p = 0.001). At the time of discharge, the eosinophilic proportion of patients was significantly higher (eosinophil %, mean ± SD; 3.06 ± 1.49 vs 2.07 ± 1.92; p = 0.001), but the activated partial thromboplastin time was significantly lower (22.34 ± 1.38 vs 24.38 ± 3.58; p = 0.01) in group 1 than in group 2. Of 14 patients with eosinophil content > 4%, 6 were in group 1 ((6/13) 46.2%), while 8 were in group 2 ((8/63) 11.9%); (p = 0.009), and all had a D-dimer level < 1 μg/mL (p = 0.03). ROC analysis for the presence of anticoagulation at subprophylactic level revealed an area under curve of 0.79 (95% CI: 0.64–0.93); p = 0.001). In conclusion; Elevated eosinophil count is related to lower anti-factor Xa activity in patients with COVID-19 receiving LMWH. The clinical significance of the subprophylactic anti-factor Xa activity should be studied in COVID-19 patients (NCT04507282).

Determination of the anticoagulant activity of low molecular weight heparins by micellar electrokinetic chromatography combined with on-column enzymatic reaction

Low molecular weight heparins (LMWHs) have largely replaced heparin for the treatment and prevention of thrombosis because of their various advantages over unfractionated heparins (UFHs) such as less bleeding, greater bioavailability, and more predictable anticoagulant effects. For special groups of patients, such as pregnant women, children, and patients with renal failure, it is necessary to monitor the anticoagulant activity of LMWHs in the blood. The traditional method used to determine the anticoagulant activity of heparin is the coagulation test. However, the results are various from different laboratories and different reagents. In contrast, the chromogenic substrate method is more accurate, sensitive and is easy to automate. Here, a method for the determination of the anticoagulant activity of LMWHs was developed by using a capillary-electrophoresis-based substrate chromogenic method. In this method, micellar electrokinetic chromatography (MEKC) was used in combination with electrophoretically mediated microanalysis to determine the anti-factor Xa (FXa) activity of LMWHs. The inhibition was measured by employing a chromogenic peptide substrate (CPS) with a p-nitroaniline (p-NA) moiety as the chromophore. The injection end of the capillary was used as a microreactor in which solutions of LMWHs, antithrombin Ⅲ (ATⅢ), FXa and CPS were successively injected and mixed by using diffusion, the transverse diffusion of laminar flow profiles and applied voltage. The reaction product p-NA was separated from unreacted CPS and sample matrix by using the MEKC mode with discontinuous background electrolyte system. The produced p-NA was baseline separated from the other components and detected at 380 nm to obtain maximum sensitivity. The amount of p-NA was inversely proportional to the activity of LMWHs in the sample. To improve the accuracy of quantification and the method repeatability of methods, nitrofurantoin (NF) was selected as the internal standard, which was added to the solution of CPS. The method was validated and used to measure a set of samples. The method is characterized by automation, good repeatability, high sensitivity, and cost-effectiveness. Additionally, the method does not interfere by the sample matrix, and thus can be used to monitor the anticoagulant activity of LMWHs in plasma.

Continuous intravenous infusion of enoxaparin controls thrombin formation more than standard subcutaneous administration in critically ill patients. A sub-study of the ENOKSI thromboprophylaxis RCT.

Standard subcutaneous low-molecular-weight heparin (LMWH) thromboprophylaxis yields low anti-factor Xa activity in patients in the intensive care unit (ICU). The aim of the study was to assess coagulation status in ICU patients randomized to receive enoxaparin thromboprophylaxis either as a standard subcutaneous bolus (SCB) or continuous intravenous infusion (CII) for 3 consecutive days after the initiation of LMWH thromboprophylaxis. Thirty-eight patients were studied by conventional coagulation variables: prothrombin fragment F 1+2 (F 1+2) representing FXa inhibition and antithrombin (AT). Additionally, 18 patients were analyzed by the thrombin generation assay-calibrated automated thrombogram (TGA-CAT). Blood samples were collected before the initiation of the LMWH thromboprophylaxis (ie, baseline), at 51 h, and at 72 h. At beginning, no differences in coagulation biomarkers were observed. The levels of F 1+2 were significantly lower at 51 and 72 h in the CII group than in the SCB group. AT levels increased during the follow-up in the CII group, unlike in the SCB group. TGA-CAT was poor in some patients overall. In a subset of patients at 51 h lag time (4.3 vs 7.5 min, respectively, P < 0.05) and time to peak (7.7 vs 14.3 min, respectively, P < 0.05) were prolonged in the SCB group. At 72 h, however, peak thrombin was lower in the CII than in the SCB group: 271 vs 356 nM, respectively (P < 0.05). Enoxaparin thromboprophylaxis administered by CII inhibited more prominently FXa and preserved better the AT level, compared with standard subcutaneous care.

Inadequate prophylactic effect of low-molecular weight heparin in critically ill COVID-19 patients.

PurposeThe aim of this study was to investigate potential markers of coagulopathy and the effects of thromboprophylaxis with low-molecular-weight heparin (LMWH) on thromboelastography (TEG) and anti-factor Xa in critically ill COVID-19 patients.Material and MethodsWe conducted a prospective study in 31 consecutive adult intensive care unit (ICU) patients. TEG with and without heparinase and anti-factor Xa analysis were performed. Standard thromboprophylaxis was given with dalteparin (75–100 IU/kg subcutaneously).ResultsFive patients (16%) had symptomatic thromboembolic events. All patients had a maximum amplitude (MA) > 65 mm and 13 (42%) had MA > 72 mm at some point during ICU stay. Anti-factor Xa activity were below the target range in 23% of the patients and above target range in 46% of patients. There was no significant correlation between dalteparin dose and anti-factor Xa activity.ConclusionsPatients with COVID-19 have hypercoagulability with high MA on TEG. The effect of LMWH on thromboembolic disease, anti-factor Xa activity and TEG was variable and could not be reliably predicted. This indicates that standard prophylactic doses of LMWH may be insufficient. Monitoring coagulation and the LMWH effect is important in patients with COVID-19 but interpreting the results in relation to risk of thromboembolic disease poses difficulties.

Effect of enoxaparin on plasma fibrin clot properties and fibrin structure in patients with acute pulmonary embolism

BackgroundLow-molecular-weight heparins (LMWHs) influence the fibrin network structure in in vitro models. There have been no reports on LMWH-induced modifications of fibrin clot characteristics and their determinants in acute pulmonary embolism (PE). AimWe investigated how enoxaparin alters fibrin clot properties in acute PE patients. MethodsClots were generated from plasma of 46 acute PE patients, aged 47-77 years treated with enoxaparin 1 mg/kg bid. Fibrin clot permeability (Ks) and clot lysis time (CLT), along with coagulation and fibrinolysis proteins were determined. Plasma fibrin clot nanostructure was assessed using scanning electron microscopy (SEM). ResultsBoth Ks and CLT were associated with anti-factor (F)Xa activity (r = 0.75, p < 0.0001 and r = −0.37, p = 0.011). Anti-FXa was positively associated with fibrin fiber diameter and the pore area, and inversely with fibrin fiber density on SEM images. Multiple regression analysis adjusted for age, body-mass index, and fibrinogen levels showed that anti-FXa activity, antithrombin activity, and FVIII activity determined Ks, while anti-FXa activity, plasminogen activator inhibitor-1 level, and presence of right ventricular dysfunction determined CLT. ConclusionsWe identified new laboratory and clinical factors contributing to prothrombotic plasma fibrin clot characteristics during enoxaparin treatment, which might help elucidate mechanisms underlying therapy failure in patients with acute PE.