Supraprophylactic Anti-Factor Xa Levels Are Associated with Major Bleeding in Neurosurgery Patients Receiving Prophylactic Enoxaparin.

BACKGROUND: Prior studies demonstrated reduced risk for venous thromboembolism (VTE) in neurosurgical patients secondary to prophylaxis with both heparin and low-molecular-weight heparin. The ability to monitor low-molecular-weight heparin by obtaining anti-factor Xa (anti-Xa) serum levels provides an opportunity to evaluate safety and efficacy. The aim of this study was to describe characteristics of patients who have anti-Xa levels outside of the goal range (0.2-0.4/0.5 IU/mL) and investigate incidence of major bleeding and VTE. METHODS: A single-center, retrospective, observational study was conducted on neurosurgical patients receiving enoxaparin for VTE prophylaxis between August 2019 and December 2020. Significance testing was conducted via Fisher exact test and independent samples t test. RESULTS: The study included 85 patients. Patients were less likely to have an anti-Xa level in the goal range if they were male, had a higher weight, or were morbidly obese. Three neuroendovascular patients (3.5%) experienced a major bleed. Serum anti-Xa levels were significantly higher in patients who experienced major bleeds compared with patients who did not (0.45 ± 0.16 IU/mL vs. 0.28 ± 0.09 IU/mL, P = 0.003). Patients with a supraprophylactic anti-Xa level (>0.5 IU/mL) were more likely to experience a major bleed (P = 0.005). One VTE event occurred: the patient experienced a pulmonary embolism with anti-Xa level at goal. CONCLUSIONS: Anti-Xa-guided enoxaparin dosing for VTE prophylaxis in neurosurgical patients may help prevent major bleeding. These data suggest that a higher anti-Xa level may predispose patients to major bleeding. Further evaluation is needed to identify the goal anti-Xa level for VTE prophylaxis in this population.

Management of Anticoagulation Therapy in Patients With Thromboembolism in the Context of Renal Dysfunction: Challenging Cases and Practical Algorithms.

BACKGROUND: Low-molecular-weight heparin is cleared through the kidneys and is commonly used for anticoagulation in the pediatric population. OBSERVATION: We present 3 challenging cases of children requiring anticoagulation in the context of acute kidney injury, nephrotic syndrome, and hemodialysis. CONCLUSIONS: A significant change in anti-factor Xa (anti-Xa) levels-used for drug monitoring-should prompt an assessment of renal function. In nephrotic syndrome, anti-Xa levels should be closely monitored when there is a change in the status of nephrotic disease activity. In hemodialysis patients, enoxaparin at once daily reduced dosing should be considered with trough and peak anti-Xa levels monitoring.

Safety and Pharmacokinetics of DS-1040 Drug-Drug Interactions With Aspirin, Clopidogrel, and Enoxaparin.

DS-1040, a novel low-molecular-weight inhibitor of activated thrombin-activatable fibrinolysis inhibitor, is under development for the treatment of thromboembolic diseases including venous thromboembolism and acute ischemic stroke. Here we describe the results of 3 studies that evaluated the safety and tolerability of DS-1040 along with the effect on DS-1040 pharmacokinetic (PK) parameters, when dosed alone or when coadministered with aspirin (NCT02071004), clopidogrel (NCT02560688), or enoxaparin in healthy subjects. Concomitant administration of single-dose DS-1040 with multiple-dose aspirin, multiple-dose clopidogrel, or single-dose enoxaparin, consistent with clinically relevant dose regimens, was safe and well tolerated with no serious treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation, bleeding-related TEAEs, and no significant changes in coagulation parameters. DS-1040 did not prolong bleeding time when administered concomitantly with aspirin or clopidogrel. In the aspirin study, DS-1040 PK was evaluated following the concomitant administration with multiple-dose aspirin, where the plasma DS-1040 exposure (peak plasma concentration [Cmax ] and area under the concentration-time curve [AUCinf ]) was to be similar to the data previously published in the first-in-human study of DS-1040 in healthy subjects. The PK parameters of DS-1040 coadministered with clopidogrel were similar to those of DS-1040 alone, with small increases in geometric means for Cmax (7%) and AUClast (9%). When coadministered with enoxaparin, the PK parameters of DS-1040 were not affected (1.1% and 1.5% decreases in geometric means for Cmax and AUClast , respectively). Therefore, concomitant administration of DS-1040 and clopidogrel or enoxaparin did not demonstrate PK drug-drug interactions.

Plasma levels of enoxaparin oligosaccharides, antifactor-Xa and thrombin generation in patients undergoing haemodialysis.

: Low molecular weight heparins are used during haemodialysis for thromboprophylaxis of the dialysis circuit, with plasma antifactor-Xa (anti-Xa) activity used as a surrogate measure for effective anticoagulation. However, this pharmacokinetic parameter does not always correlate with pharmacodynamic effects in patients. The aim of this study was to investigate the relationship between actual plasma levels of the low molecular weight heparins enoxaparin, anti-Xa activity, and global coagulation measurement of thrombin generation during haemodialysis. Blood was analysed from 16 adult patients with end-stage kidney disease at 0, 2, 4 h, and at completion of 31 dialysis sessions where single fixed doses of 20 (n = 3), 40 (n = 16), 60 (n = 6), or 80 (n = 6) mg of enoxaparin (equating to 0.23-1.07 mg/kg) were used as thromboprophylaxis. Plasma enoxaparin oligosaccharides [degree of polymerization (dp)6-dp16] were measured by high-performance size exclusion chromatography, anti-Xa activity by colourimetric assay, and thrombin generation by calibrated automated thrombogram. Plasma enoxaparin fragments were undetectable at the beginning of each dialysis, peaked at 2 h to levels that correlated with dose (r = 0.68, P < 0.001) then remained relatively stable. In contrast, therapeutic anti-Xa levels achieved at 2 h in 18 cases (58%) quickly dropped to only six cases (19%) at the end of dialysis, by which time thrombin generation had also recovered in 81% of patients. Statistical modelling revealed a threshold value of anti-Xa at 0.53 IU/ml that supressed thrombin generation to 15.28% of baseline (P < 0.001). Despite loss of anticoagulant activity in the majority of patients, plasma levels of enoxaparin oligosaccharides remained detectable and relatively unchanged throughout dialysis.

Low-molecular-weight heparin adherence and effects on survival within a randomised phase III lung cancer trial (RASTEN).

BACKGROUND: Coagulation activation is a hallmark of cancer, and anticoagulants have shown tumour-inhibiting properties. However, recent trials have failed to demonstrate improved survival with low-molecular-weight heparin (LMWH) in cancer populations. This has raised the question of suboptimal adherence as a possible explanation for the lack of benefit. Still, there is no standardised method to directly monitor LMWH in patient plasma. Here, we directly determine LMWH levels in patients using the Heparin Red assay to objectively assess adherence and how this associates with the patient outcome in the RASTEN trial. METHODS: RASTEN is a multicentre, randomised phase III trial investigating if the addition of LMWH to standard therapy can improve survival in small-cell lung cancer. LMWH was measured in plasma (N = 258) by the Heparin Red assay and compared with the anti-factor Xa (anti-FXa) activity assay. RESULTS: Both methods could differentiate patients in the LMWH arm from the control arm and patients receiving therapeutic LMWH owing to thrombosis. Receiver Operating Characteristic (ROC) analysis yielded adherence rates of 85% for anti-FXa and 68% for Heparin Red. No survival benefits were found in the adherent subgroup compared with the control arm (hazard ratio [HR]: 1.26; 95% confidence interval [CI]: 0.95-1.67; P = 0.105 and HR: 1.19; 95% CI: 0.89-1.60; P = 0.248 for anti-FXa and Heparin Red, respectively). Heparin Red could define patients with high probability of adherence to LMWH treatment, which warrants prospective studies for further validation. Our finding that the LMWH-adherent subpopulation did not show improved survival excludes that the negative outcome of RASTEN was due to poor adherence.

Retrospective comparison of two enoxaparin dosing and monitoring protocols at a pediatric hospital.

PURPOSE: The study analyzes the effectiveness and safety of a higher than standard enoxaparin dosing protocol implemented for pediatric patients requiring initiation of therapeutic anticoagulation. METHODS: A retrospective review of 2 enoxaparin dosing and monitoring protocols was performed. The standard protocol used 1.5 mg/kg/dose (in patients <3 months of age) and 1 mg/kg/dose (in patients ≥3 months of age) with anti-Xa monitoring following the first dose. The high-dose protocol was implemented at 1.7 mg/kg/dose (in patients <3 months of age), 1.5 mg/kg/dose (in patients 3 through 11 months of age), 1.2 mg/kg/dose (in patients 1 through 4 years of age), and 1.1 mg/kg/dose (in patients 5 through 17 years of age), with anti-Xa monitoring after the second dose. Primary outcomes were number of dosing changes prior to and time to first target anti-Xa level. Secondary outcomes included percentage of patients with anti-Xa levels above target level. RESULTS: The median number of dose changes required to achieve a target anti-Xa level was 1 (interquartile range [IQR], 0-1.5) and 0 (IQR, 0-1) for the standard-dose (n = 87) and high-dose groups (n = 132) (p = 0.17), respectively. The median number of dose adjustments to achieve target anti-Xa levels in the 3 through 11 months of age subgroup declined from 2 (IQR, 1-3.25) to 0 (IQR, 0-1) in the standard- versus high-dose groups, respectively (p < 0.01). No difference was seen in other age subgroups. Patients with above-target levels did not differ statistically between groups. CONCLUSION: Initiating enoxaparin at higher doses in pediatric patients may result in fewer dosing changes than standard dosing. Benefit was demonstrated for the 3-11 months of age high-dose subgroup. Across all groups, the high-dose strategy was safe and did not result in a statistically significant increase in above-target levels.

Enoxaparin Population Pharmacokinetics in the First Year of Life.

AIMS: Enoxaparin dosing requirements in the first year of life can be highly variable. Characterization of pharmacokinetics in this patient population can assist in dosing. METHODS: Patients less than 1 year postnatal age who received enoxaparin and had an anti-factor Xa activity level drawn as inpatients were identified through the pharmacy database over a 5-year period. Patients on renal replacement therapy or with hyperbilirubinemia were excluded. Data collection included demographic variables, indication for enoxaparin, enoxaparin doses, anti-factor Xa activity levels, serum creatinine, hemoglobin, hematocrit, platelet count, and urine output over the previous 24 hours. Population pharmacokinetic analysis was performed with NONMEM. RESULTS: A total of 182 patients [male 50%, median 100 days postnatal age (range: 4-353 days)] met the study criteria. Patients received median 22 doses (range: 1-526) at a mean starting dose of 1.38 ± 0.43 mg/kg with median 5 (range: 1-56) anti-factor Xa activity levels measured. A 1-compartment proportional and additive error model best fits the data. Allometrically scaled weight significantly decreased the objective function value, as did serum creatinine on clearance, and postmenstrual age (PMA) on volume of distribution. When evaluated graphically, dosing based on PMA appeared to have less variability as compared to postnatal age-based dosing. CONCLUSIONS: Dosing of enoxaparin in infants younger than 1 year should incorporate PMA.

Treatment of suspected pulmonary embolism in a morbidly obese patient.

PURPOSE: A case highlighting challenges with enoxaparin dosage and monitoring in obese patients is presented. SUMMARY: A morbidly obese 22-year-old Caucasian female (height, 168 cm; weight, 322 kg; body mass index [BMI], 114 kg/m2) who presented to the emergency department with acute-onset dyspnea and hypoxia was empirically initiated on enoxaparin for suspected pulmonary embolism at the institution's standard maximum dosage (160 mg subcutaneously every 12 hours). On hospital day 2, a peak anti-factor Xa (anti-Xa) level of 0.4 IU/mL was documented about 4 hours after the fourth enoxaparin dose. On hospital day 3, the enoxaparin dose was increased to 200 mg, a dose equivalent to 0.62 mg per kilogram of actual body weight (ABW), much lower than the guideline-recommended dose for venous thromboembolism prophylaxis (1 mg/kg). Four hours after her third 200-mg dose of enoxaparin, the patient had an anti-Xa value of 0.64 IU/mL (goal range, 0.5-1.1 IU/mL), with no evidence of bleeding or other adverse drug events. Follow-up anti-Xa testing on hospital day 4 yielded a value of 0.78 IU/mL. The case highlights the need for research to better delineate strategies for enoxaparin dosage and monitoring in the context of extreme obesity. CONCLUSION: A female patient with a BMI of 114 kg/m2 was safely and effectively treated using an initial twice-daily dose of enoxaparin less than the recommended 1-mg/kg dose based on ABW. Dosage adjustments were made according to anti-Xa levels, and no adverse drug reactions were noted.

Individualised medicine: why we need Bayesian dosing.

Individualised drug dosing has been shown to improve patient outcomes and reduce adverse drug events. One method of individualised medicine is the Bayesian approach, which uses prior information about how the population responds to therapy, to inform clinicians about how a specific individual is responding to their current therapy. This information is then used to make changes to the dose. Studies using a Bayesian approach to adjust drug dosing have shown that clinicians are able to achieve a therapeutic range quicker than standard practice. If concentration is related to a pharmacodynamic end-point, this means that the drug will be more effective, and the side-effects will be minimised. Unfortunately, the software options to assist with Bayesian dosing in Australia are limited. The aims of this article are to demystify the concepts of Bayesian dosing, set the context of the Bayesian approach using reference to other dosing strategies and discuss its benefits over current dosing methods for a number of drugs. The article is targeted to medical and pharmacy clinicians, and there is a practical clinical case to demonstrate how this method could be used in everyday clinical practice.

Residual Enoxaparin Activity, Anti-Xa Levels, and Concerns About the American Society of Regional Anesthesia and Pain Medicine Anticoagulation Guidelines.

Currently, the American Society of Regional Anesthesia and Pain Medicine (ASRA) anticoagulation guidelines recommend that before the performance of a neuraxial procedure a minimum of 24 hours should elapse following a treatment dose of enoxaparin (1 mg/kg twice daily or 1.5 mg/kg once daily). The guidelines have since their inception also consistently recommended against the routine use of anti-Xa level monitoring for patients receiving enoxaparin. However, we noted in our clinical practice that anti-Xa levels were frequently still elevated despite patients meeting the time-based recommendation for treatment dose enoxaparin. To further investigate the possibility that residual anticoagulant activity may persist longer than 24 hours after a treatment dose of enoxaparin, we assessed anti-Xa level activity in patients presenting for elective surgery. Despite nearly universal compliance with ASRA's anticoagulation guidelines (1 sample was drawn at 23.25 hours), anti-Xa activity was found to be elevated in 11 of 19 patients. While 10 patients had an anti-Xa level within the peak prophylactic range (0.2-0.5 IU/mL), 1 patient's level was found to still be in the peak therapeutic range (0.5-1.0 IU/mL). These findings suggest that significant anticoagulant activity may persist longer than previously appreciated after the last treatment dose of enoxaparin and that the current time-based ASRA recommendation may not be conservative enough. Further research is needed to delineate the level of anti-Xa activity below which it is likely safe to proceed with a neuraxial procedure, but it may be time to reconsider the utility of anti-Xa level monitoring when it is available.

A single test to assay warfarin, dabigatran, rivaroxaban, apixaban, unfractionated heparin, and enoxaparin in plasma.

UNLABELLED: Essentials Simple and fast assaying of different anticoagulants (ACs) is useful in emergent situations. We used highly diluted prothrombin time (dPT) or highly diluted Fiix-PT (dFiix-PT) to assay ACs. Both tests could quantify target specific anticoagulants and warfarin anticoagulation. Improved results were consistently observed with the dFiix-PT compared with the dPT. SUMMARY: Background Assaying anticoagulants is useful in emergency situations or before surgery. Different specific assays are currently needed depending on the anticoagulant. Objectives We hypothesized that levels of warfarin, dabigatran, rivaroxaban, apixaban, and heparins could be measured with use of the diluted prothrombin time (dPT) and diluted Fiix-PT (dFiix-PT), using highly diluted thromboplastin (TP). The latter test is affected only by reduced levels of active factors II and X but corrects test plasma for other deficiencies Methods Increasing TP dilutions were used to identify suitable dilutions to measure dabigatran, rivaroxaban, apixaban, unfractionated heparin (UFH), and enoxaparin. Calibrators containing known amounts of direct oral anticoagulants (DOACs) were used to make standard curves. Citrated plasma samples were obtained from patients taking warfarin or DOACs with known drug concentrations as determined by specific assays. Results The dFiix-PT at a TP dilution of 1:1156 could be used to measure all of the drugs tested at therapeutic concentrations except for fondaparinux. The dPT achieved the same but required two TP dilutions (1:750 and 1:300). The warfarin effect could be assessed by using dFiix-PT at 1:1156 with a PT ratio identical to the international normalized ratio. Six different TPs yielded similar results, but two were less sensitive. Dabigatran, rivaroxaban, and apixaban could be accurately measured in patient samples using both dilute PT assays, but a better correlation was consistently observed between the dFiix-PT and specific assays than with the dPT. Conclusion The dFiix-PT using a single dilution of TP may be suitable to assess the anticoagulant effects of warfarin, dabigatran, rivaroxaban, apixaban, heparin, and enoxaparin.

Investigation of an anti-activated factor X (anti-Xa) assay for the quantification of enoxaparin in human plasma.

OBJECTIVES: Enoxaparin is a low-molecular weight heparin (LMWH) widely used for the treatment of thrombosis and measured through a chromogenic assay (anti-Xa). The aim of this project was to investigate the impact of patient and sampling sources of variation on an anti-Xa assay for enoxaparin. METHODS: COATEST was used in accordance with manufacturer's instructions. A standard curve of LMWH concentration (0.1-1.0 IU/ml) was prepared. The shelf-life and freeze-thaw stability of Xa over a 6-month period were investigated. The effects of blood sample haemolysis and plasma antithrombin-III (AT) concentration were examined. KEY FINDINGS: The standard curve performed well with high accuracy (average bias of 8.1%) and precision (average CV of 2.8%). The shelf-life of Xa once reconstituted could be extended from 1 month to 4 months if aliquots were frozen at -20°C. Some loss of anti-Xa activity was observed on freeze-thawing (bias up to 25%). Haemolysis of 2.5% was found to interfere with the assay. AT level as low as 50% did not affect assay accuracy. CONCLUSIONS: Xa standards when frozen immediately after reconstitution could be used for the following 4 months. Haemolysis interferes with the assay, but a 50% reduction in AT does not significantly affect the assay result.

Development and evaluation of PEGylated Enoxaparin: a novel approach for enhanced anti-Xa activity.

Enoxaparin (ENX) is one of the most widely prescribed low molecular weight heparin inprophylaxis and treatment of venous thromboembolism. In this study, Enoxaparin-PEG conjugate (P-ENX) was synthesized from Enoxaparin and polyethylene glycol (PEG) and evaluated for its potential for extended duration of action. The esterification of the carboxyl groups of the drug moiety with the hydroxyl groups of mPEG-2000 was done by employing carbodiimide coupling chemistry. P-ENX conjugate was purified by dialysis and characterized by Fourier transform infrared spectroscopy (FTIR), Proton-Nuclear magnetic resonance ((1)H NMR) and matrix-assisted laser desorption/ionization (MALDI) mass analysis techniques. FTIR analysis revealed frequency of the carbonyl group in accord with ester linkage formation between the drug and the PEG moiety. (1)H NMR of the conjugate showed significant change in the chemical shift further indicative of ENX and PEG chemical interaction. In MALDI spectra, small peaks at 12,907 and 16,137 m/z confirmed the probability of conjugation of ENX and PEG. P-ENX exhibited considerable enhancement in anti-Xa activity (by three-folds) in comparison to free ENX. Further, an increase in AUC (over four-folds) was observed in P-ENX. Thus, PEGylation of ENX is a novel approach for extended and enhanced activity of ENX with a potential for decreased dosing frequency.

Rapid point-of-care assay of enoxaparin anticoagulant efficacy in whole blood.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 °C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 °C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. (2-4) The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Leveraging physiological data from literature into a pharmacokinetic model to support informative clinical study design in pregnant women.

PURPOSE: Physiological changes during pregnancy can effect pharmacokinetic (PK) parameters, which may lead to altered dose requirements. We aimed to leverage literature-based physiological changes during pregnancy into a PK model and compare its performance to a published reference model in pregnant women and to use the literature-based model to determine informative PK sampling times for a clinical study that aims to quantify the PK of enoxaparin throughout pregnancy. METHODS: Changes in total body water (BW) and creatinine clearance (CRCL) during pregnancy were described using regression models. BW and CRCL were linked to a PK model of enoxaparin in non-pregnant women. Performance of the literature-based PK model was compared to a previously published empirical reference model. D-optimal sampling times were determined and evaluated for literature-based and reference models. RESULTS: The literature-based model adequately predicted anti-Xa plasma concentrations when compared to reference model predictions. An informative sampling design was successfully developed, with parameters expected with good precision (RSE < 36.4%). CONCLUSION: A literature-based model describing enoxaparin PK during pregnancy was developed and evaluated. The modelling framework could be used to support development of informative designs in pregnancy when prior models are unavailable.

Comparison of the anticoagulant response of a novel fluorogenic anti-FXa assay with two commercial anti-FXa chromogenic assays.

INTRODUCTION: Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the 'gold standard' assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity. MATERIALS AND METHODS: Commercial plasmas were spiked with pharmacologically relevant concentrations (0-1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7µM fluorogenic substrate, in addition to 6µl of 100mM CaCl(2) and 44µl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed. RESULTS: The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0-1 U/ml with CVs of<5% for UFH and tinzaparin and CVs<9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits. CONCLUSIONS: This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Determination of enoxaparin with rotational thrombelastometry using the prothrombinase-induced clotting time reagent.

Drug monitoring of low molecular weight heparin is generally not recommended, but could be reasonable in critically ill patients, whose risk for bleeding or thrombosis shows a high interpatient variability. Anti-Xa assays are not available around the clock even in central hospitals, whereas rotational thrombelastometry (ROTEM) becomes increasingly used at the bedside. Prothrombinase-induced clotting time (PiCT) reagent allows determination of factor Xa-inhibition in plasma. The aim of our study was to evaluate enoxaparin determination in whole blood with the ROTEM using specific test modifications, including PiCT. After ethics committee's approval, citrated whole blood obtained from overall 16 healthy volunteers was incubated with enoxaparin at 16 different anti-Xa concentrations. Main endpoint was the clotting time (CT) in ROTEM representing initial activation of clot formation. CT was determined in the new PiCT-ROTEM test, in a low-tissue factor-activated modification (LowTF-ROTEM) as well as in the commercially available heparin-sensitive ROTEM assays (HEPTEM and INTEM). In the absence of enoxaparin, CT values were 168.6 +/- 6.1 s (PiCT-ROTEM), 247.3 +/- 18.6 s (LowTF-ROTEM), and -6.2 +/- 7.9 s (INTEM-HEPTEM). A linear dependency (P < 0.01) between anti-Xa concentration and CT was found for PiCT-ROTEM, LowTF-ROTEM, and for INTEM-HEPTEM with correlation coefficients of 0.93 for PiCT-ROTEM, 0.94 for LowTF-ROTEM, and 0.81 for INTEM-HEPTEM. This in-vitro experiment demonstrates a strong correlation between enoxaparin anti-Xa concentrations and specific ROTEM tests. These promising assays should be further evaluated for monitoring anticoagulation in high-risk patients in clinical studies.

Stability of plasma anti-Xa activity in low-molecular-weight heparin monitoring.

Low-molecular-weight heparin (LMWH) is now the standard of care for prophylaxis and treatment of thromboembolic disorders. Only cases with renal failure, morbid obesity or extreme age require anti-Xa monitoring to assure the therapeutic level achievement. Because of infrequent requests, the test is usually sent to the reference laboratories and specimen handling may be delayed. Because LMWHs can be kept at ambient temperature for several days, we proposed that anti-Xa levels in plasma samples are similarly steady. Patients' plasma that was requested for anti-Xa activity was left at room temperature to repeat the test 24 hours later and compare with the result of immediate assay. The study included 86 fresh specimens from 56 participants. All patients received enoxaparin with anti-Xa levels ranging from 0.1 to 2.5 U/mL. Notably, anti-Xa activities significantly rose on the second occasions (P = 8.4 x 10(-10)). The mean change of anti-Xa was +0.15 +/- 0.21 U/mL (+24.9% +/- 37.4%). Children (age <15 years) showed more marked alterations than adults (+40.9% vs. +18.2%, P = .008). There was no statistical difference in the degrees of changes between sexes and diagnoses. The data suggest that specimens sent for anti-Xa require prompt handling to prevent falsely elevated values. This observation is new and future research is needed to find the mechanism of this alteration.