Anti-factor Xa Assay Research Articles

The use of activated partial thromboplastin time versus antifactor Xa assay in monitoring continuous unfractionated heparin infusion therapy in obstetric intensive care unit

BackgroundUnfractionated heparin (UFH) infusion therapy needs accurate tight control to prevent overanticoagulation that may result in hemorrhagic complications and also to prevent sub therapeutic level that may result in thrombotic complications. The aim of this study was to compare the most popular monitoring tool activated aPTT versus antifactor Xa to reach accurate monitor to UFH therapy in critically ill pregnant females.ResultsTime to reach therapeutic level as well as total heparin dose required to reach this therapeutic level were much lower in the anti-Xa group when compared to aPTT group.ConclusionThe use of anti-Xa-based protocol to monitor UFH infusion therapy resulted in better therapeutic control as it resulted in earlier achievement of therapeutic level and lower heparin dose requitments.

Significant Reduction of Pulmonary Embolism in Orthopaedic Trauma Patients

To report results of a protocol to lessen incidence of pulmonary embolism (PE) among orthopaedic trauma patients. Retrospective review. Level 1 trauma center. Orthopaedic trauma inpatients were included in the study. On arrival, an orthopaedic trauma patient's PE risk is calculated using a previously developed tool. If possible, patients at high risk are given their first dose of enoxaparin before leaving the emergency room. If other injuries preclude enoxaparin, then chemoprophylaxis is held for 24 hours. Twenty-four hours after arrival, the patient's ability to receive enoxaparin is reassessed. If possible, enoxaparin is started, with dosing twice a day. If enoxaparin is still contraindicated, a removable inferior vena cava filter is placed. Adequacy of enoxaparin dosing is tested using anti-factor Xa assay, drawn 4 hours after the third dose of enoxaparin. If the anti-factor Xa result is less than 0.2 IU/mL, a removable inferior vena cava filter is placed. If the result is 0.2-0.5 IU/mL, enoxaparin dosing is continued. If greater than 0.5 IU/mL, the dose of enoxaparin is reduced. The main outcome measure was rate of PE. From September 1, 2015 to December 31, 2015, our hospital admitted 420 orthopaedic trauma patients. Fifty-one patients were classed as high risk for PE. In September through December 2015, 9 sustained PE, 1 of which was fatal. From September 1, 2016 to December 31, 2016, our hospital admitted 368 orthopaedic trauma patients with comparable age and Injury Severity Score to 2015. Forty patients were at high risk for PE, 1 sustained a nonfatal PE. PE incidence from September to December 2016 was significantly lower than in 2015 (P = 0.02). Overall, 26 patients managed under the new protocol had IVCFs placed, 21 had their filters removed, and 3 died with filters in place. There were no complications during filter placement or removal. One patient had hemorrhage felt to be attributable to enoxaparin. Our protocol emphasizes more robust enoxaparin dosing, and more frequent use of IVCF, but only among those at high risk. We lessened the incidence of PE, with a low complication rate. Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Quantification of apixaban in human plasma using ultra performance liquid chromatography coupled with tandem mass spectrometry.

Apixaban, an inhibitor of direct factor Xa, is used for the treatment of venous thromboembolic events or prevention of stroke. Unlike many other anticoagulant agents, it does not need periodic monitoring. However, monitoring is still required to determine the risk of bleeding due to overdose or surgery. Usually, apixaban concentrations are indirectly quantified using an anti-factor Xa assay. However, this method has a relatively narrow analytical concentration range, poor selectivity, and requires an external calibrator. Therefore, the goal of current study was to establish an analytical method for determining plasma levels of apixaban using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To this end, apixaban was separated using 2.5 mM ammonium formate (pH 3.0) (A) and 100% methanol containing 0.1% formic acid (B) using the gradient method with a Thermo hypersil GOLD column. The mass detector condition was optimized using the electrospray ionization (ESI) positive mode for apixaban quantification. The developed method showed sufficient linearity (coefficient of determination [r2 ≥ 0.997]) at calibration curve ranges. The percentage (%) changes in accuracy, precision, and all stability tests were within 15% of the nominal concentration. Apixaban concentration in plasma from healthy volunteers was quantified using the developed method. The mean maximum plasma concentration (Cmax) was 371.57 ng/mL, and the median time to achieve the Cmax (Tmax) was 4 h after administration of 10 mg apixaban alone. Although the results showed low extraction efficiency (~16%), the reproducibility (% change was within 15% of nominal concentration) was reliable. Therefore, the developed method could be used for clinical pharmacokinetic studies.

How and when to measure anticoagulant effects of direct oral anticoagulants? Practical issues.

Direct oral anticoagulants (DOACs) do not require dose adjustment based on laboratory testing. However, it might be necessary to measure their plasma concentrations in the following specific situations: 1) before thrombolytic therapy in patients with stroke; 2) before surgery or invasive procedure; 3) in case of adverse events (thrombosis or hemorrhage); 4) when immediate reversal of anticoagulation is needed; 5) in patients with extreme body weight; 6) when administering additional drugs potentially interfering with DOACs; and 7) when overdosage is suspected regardless of concomitant bleeding. Basic coagulation tests, such as prothrombin and activated partial thromboplastin time, should not be used as standalone tests to assess the levels of anticoagulation as they are not specific for DOACs and their results are dependent on the type of reagent used for testing. Plasma DOAC concentrations should be assessed by dedicated tests: dilute thrombin time or ecarin tests (for dabigatran) or anti-factor Xa assays (for anti-factor Xa inhibitors). Dedicated tests should be calibrated against their respective plasma calibrators at certified DOAC concentrations and results should be expressed as ng/ml. Caution should be exerted when interpreting the results of the most common hemostatic parameters such as antithrombin, proteins C and S, lupus anticoagulant, or individual coagulation factors, as they may be strongly affected by the presence of a DOAC. Whenever possible, these parameters should be measured 4 to 5 days after discontinuation of DOAC anticoagulation.

Assessment of Heparin Anticoagulation Measured Using i-STAT and Hemochron Activated Clotting Time

Adequate anticoagulation, measured using activated clotting time (ACT), is important during vascular and cardiac surgeries. Unfractionated heparin is the most common anticoagulant used. The purpose of this analysis was to compare the i-STAT ACT (iACT) to the Hemochron ACT (hACT), both of which were then compared to anti-factor Xa (anti-Xa) assay, a representation of heparin level and activity. Prospective study. Tertiary care cardiovascular center. Eleven consecutive elective adult cardiac surgical patients. Prior to cardiopulmonary bypass, ACTs were measured using i-STAT and Hemochron technologies and compared to each other and to anti-Xa assay prior to and during a cumulative administration of heparin. Data were compared using bias analyses. Heparin (300 U/kg) was administered in quarterly doses. Coagulation labs were collected prior to and 3 minutes after each quarterly dose of heparin. The baseline ACTs for i-STAT and Hemochron were 147 and 142 seconds, respectively. A significant association was found between iACT and hACT (p = 0.002). The iACT measurements underestimated hACT at ACT levels >180 seconds or anti-Xa levels >0.75 U/mL. No significant difference was found between ACT data at anti-Xa levels <0.5 U/mL. There was a good association between the iACT and hACT; however, the 2 tests are not equivalent. Overall, the iACT underestimated the hACT. Agreement between the ACT technologies was good at lower ACTs and anti-Xa levels, but declined with an anti-Xa >0.75 U/mL.

Laboratory measurement of apixaban using anti-factor Xa assays in acute ischemic stroke patients with non-valvular atrial fibrillation.

Apixaban is effective and safe for preventing stroke, and its usage has increased exponentially in recent years. However, data concerning the therapeutic range of apixaban is limited. This study determined the trough and peak levels of apixaban-specific anti-factor Xa activity (AFXaA) in acute ischemic stroke patients with non-valvular atrial fibrillation (NVAF) in Korea. The study included 85 patients who received apixaban. Blood samples were taken to measure the trough and peak levels of AFXaA using a chromogenic anti-factor assay, as well as prothrombin time (PT) and activated partial thromboplastin time (aPTT). We also reviewed complications such as major bleeding of patients treated with apixaban. In patients given a 5.0-mg apixaban dose, the median trough and peak levels of AFXaA were 104.5 and 202.0ng/mL. In patients given a 2.5-mg apixaban dose, the median trough and peak AFXaA levels were 76.0 and 151.0ng/mL. The PT showed a positive correlation with increased AFXaA activity at both levels (Trough R = 0.486, Peak R = 0.592), but the aPTT had no relationship with AFXaA activity at both levels (Trough R = 0.181, Peak R = 0.129). Two cases with intracranial bleeding belonged to the highest AFXaA quartile (Trough, p = 0.176; Peak, p = 0.053). In conclusion, we determined the trough and peak levels of AFXaA in patients with NVAF while being treated with the apixaban in Korea. Our results could be used as a starting point when setting the reference ranges for laboratories using anti-Xa assay. Large-scale studies are needed to establish the reference range for AFXaA in patients with NVAF.

Anticoagulant properties of a high molecular weight polysaccharide fraction (1000RS) of the ascidian Microcosmus exasperatus

Aims: The anticoagulant effect and cytotoxicity of a high molecular weight polysaccharide fraction (1000RS) obtained from the tunic of the ascidia Microcosmus exasperatus were evaluated. Methods: Anticoagulant properties of 1000RS was evaluated by activated Partial Thromboplastin Time (aPTT), Thrombin Time (TT), Prothrombin Time (PT), anti-factor Xa and lupic anticoagulant (dRVVT) assays. Cytotoxicity was tested on murine hematopoietic cells using MTT assay. Results: This galactose rich fraction showed to be a potential anticoagulant due to its inhibitory effect on the intrinsic coagulation pathway. At the same time, anticoagulant doses of this fraction have no effect on cellular viability, which means that it can be used as a therapeutic agent. Conclusion: In vitro anticoagulant effect of 1000RS occurs at innocuous doses, however, it still need to be tested using in vivo models and its cytotoxicity evaluated in normal human cell lines.

Intravenous Thrombolysis in Patients with Stroke Taking Rivaroxaban Using Drug Specific Plasma Levels: Experience with a Standard Operation Procedure in Clinical Practice.

Background and Purpose Standard operating procedures (SOP) incorporating plasma levels of rivaroxaban might be helpful in selecting patients with acute ischemic stroke taking rivaroxaban suitable for IVthrombolysis (IVT) or endovascular treatment (EVT).MethodsThis was a single-center explorative analysis using data from the Novel-Oral-Anticoagulants-in-Stroke-Patients-registry (clinicaltrials.gov:NCT02353585) including acute stroke patients taking rivaroxaban (September 2012 to November 2016). The SOP included recommendation, consideration, and avoidance of IVT if rivaroxaban plasma levels were <20 ng/mL, 20‒100 ng/mL, and >100 ng/mL, respectively, measured with a calibrated anti-factor Xa assay. Patients with intracranial artery occlusion were recommended IVT+EVT or EVT alone if plasma levels were ≤100 ng/mL or >100 ng/mL, respectively. We evaluated the frequency of IVT/EVT, door-to-needle-time (DNT), and symptomatic intracranial or major extracranial hemorrhage.Results Among 114 acute stroke patients taking rivaroxaban, 68 were otherwise eligible for IVT/EVT of whom 63 had plasma levels measured (median age 81 years, median baseline National Institutes of Health Stroke Scale 6). Median rivaroxaban plasma level was 96 ng/mL (inter quartile range [IQR] 18‒259 ng/mL) and time since last intake 11 hours (IQR 4.5‒18.5 hours). Twenty-two patients (35%) received IVT/EVT (IVT n=15, IVT+EVT n=3, EVT n=4) based on SOP. Median DNT was 37 (IQR 30‒60) minutes. None of the 31 patients with plasma levels >100 ng/mL received IVT. Among 14 patients with plasma levels ≤100 ng/mL, the main reason to withhold IVT was minor stroke (n=10). No symptomatic intracranial or major extracranial bleeding occurred after treatment.Conclusions Determination of rivaroxaban plasma levels enabled IVT or EVT in one-third of patients taking rivaroxaban who would otherwise be ineligible for acute treatment. The absence of major bleeding in our pilot series justifies future studies of this approach.

Influence of Direct Oral Anticoagulants on Anti-Factor Xa Measurements Utilized for Monitoring Heparin.

Unanticipated drug-laboratory interactions may occur between direct oral anticoagulants (DOACs) and anti-factor Xa (AXA) levels used to monitor parenteral heparin infusions. Characterization of the extent and duration of DOAC effect on AXA levels may reduce complications with transition to heparin infusions. To evaluate the impact of oral factor Xa inhibitors on AXA levels with and without concurrent heparin. This retrospective descriptive study was approved by institutional review board waiver and included patients with AXA levels drawn on a heparin calibrated assay who had received a factor Xa inhibitor. Participants were divided on the basis of whether AXA levels were drawn with concurrent parenteral heparin. If transitioned to heparin, number of AXA draws required until AXA level was within therapeutic range was recorded. A total of 50 patients (60% rivaroxaban, 40% apixaban) met inclusion criteria. When AXA levels were drawn within 12 hours of apixaban without concurrent heparin (n = 7), 71% were greater than 1 IU/mL, and 29% were below suggested trough levels (0.7-1.1 IU/mL). For AXA levels drawn within 24 hours of rivaroxaban without concurrent heparin (n = 11), 55% were greater than 1 IU/mL, 9% were within suggested trough (0.6-1 IU/mL), and 36% were below 0.6 IU/mL. In patients (n = 28) who were initiated on heparin infusion prior to AXA monitoring, administration of the DOAC within the prior 72 hours resulted in supratherapeutic initial AXA levels 69% of the time. DOACs may cause elevations in heparin-calibrated AXA assays; this creates problematic challenges in using the AXA level to optimize heparin management.

The role of new oral anticoagulants in orthopaedics: an update of recent evidence.

Rivaroxaban, dabigatran, apixaban and edoxaban are the four available new oral anticoagulants (NOAC) which are currently approved for venous thromboembolism prophylaxis after total hip and knee replacement. Large phase 3 and phase 4 studies comparing NOAC with low molecular weight heparins have shown similar results regarding the efficacy and safety of these two categories of anticoagulants. Management of bleeding complications is a matter of great significance. Three reversal agents have been developed: idarucizumab, andexanet alfa and ciraparantag. Idarucizumab is now commercially available. Regarding the perioperative management of NOAC, two main scientific groups have published their own recommendations. The European Heart Rhythm Association recommends 48-h period of stoppage preoperatively for factor Xa inhibitors and at least 3 or 4days for dabigatran, while the French Study Group on Thrombosis and Haemostasis recommends 5-day discontinuation for all NOAC. Conventional clot tests can only be used as rough indicators for laboratory assessment of the activity of NOAC. Specific laboratory tests have been developed for more accurate measurements of NOAC blood levels, including a dilute thrombin time test (Hemoclot test) and the ecarin clot test for dabigatran and chromogenic anti-factor Xa assays for direct factor Xa inhibitors. Due to the beneficial properties of NOAC, these drugs are gaining ground in daily orthopaedic practice, and many studies are being conducted in order to extend the indications of these anticoagulants agents.

Determination of non-Vitamin K oral anticoagulant (NOAC) effects using a new-generation thrombelastography TEG 6s system.

Non vitamin K oral anticoagulants (NOACs) do not require regular monitoring but information about their pharmacodynamic effect may be importantin situations like trauma, stroke oremergent surgery. Currently, no standardized point-of-care test is available to evaluate the anticoagulant effects of NOACs. We evaluated the anticoagulant effect of NOACs with the next generation point-of-care TEG assay (TEG® 6S) based on a fully-automated thrombelastography system. We used two TEG® 6S assays, the DTI assay and Anti-Factor Xa (AFXa) assay, to detect anticoagulant effects and classify NOACs. Blood from healthy volunteers (n = 26) was used to obtain a baseline reference range. Data derived from patients on factor Xa inhibitors (FXi) (rivaroxaban and apixaban) (n = 39), and direct thrombin inhibitors (DTIs) (dabigatran) (n = 25) were compared against the reference range for detection of drug effect and drug classification. TEG®6s R-time highly correlated to each NOAC. Presence of NOACs caused elongation of R-time on the AFXa assay compared to the reference range (4.3 ± 1.7 vs. 1.3 ± 0.3min. for FXi, p < 0.001 and 3.5 ± 1.2 vs. 1.3 ± 0.3min. for DTI, p < 0.001). R-time on the DTI assay was elongated only in presence of a DTI (3.4 ± 1.0 vs. 1.5 ± 0.2min, p < 0.001). The cutoff for detection of a DTI effect was an R time of 1.9min and for anti-Xa effect was 1.95min. For detection of NOAC therapy, there was ≥92% sensitivity and ≥95% specificity. The automated TEG®6s NOAC assay may be an effective tool to identify an anticoagulant effect from NOAC therapy and facilitate care of patients with bleeding or at risk of bleeding in the event of needing emergency surgery.

A fluorescent probe assay (Heparin Red) for direct detection of heparins in human plasma.

Heparins are widely used anticoagulant drugs. The current monitoring practice for heparin in plasma, such as the chromogenic anti-factor Xa assay, relies on heparin-triggered activation of antithrombin, an inhibitor of coagulation proteases. Such assays are not applicable to the detection of non-anticoagulant heparins, an emerging class of drug candidates for therapeutic applications unrelated to anticlotting activity. This study describes the application of a commercially available fluorescent probe assay (Heparin Red) for the direct and sensitive detection of the "chemical" heparin in plasma, independent of any anticoagulant activity. The quantification range is about 0-5μg/mL for both unfractionated heparin (corresponding to 0-1IU/mL) and the low molecular weight heparin enoxaparin. The Heparin Red assay is of particular value for the quantification of non-anticoagulant heparins, as exemplified by the low molecular weight heparin derivative tafoxiparin and a N-desulfated-N-reacetylated heparin. Heparin octa- and decasaccharides are also detected. Graphical abstract Heparin quantification in plasma by mixing the sample with the Heparin Red reagent and fluorescence readout.

Heparin-induced thrombocytopenia in cardiac surgery and critically ill patients.

Thrombocytopenia as well as anti-platelet factor 4/heparin (PF4/H) antibodies are common in cardiac surgery patients and those treated in the intensive care unit. In contrast, heparin-induced thrombocytopenia (HIT) is uncommon in these populations (~1 % and ~0.5 %, respectively). A stepwise approach where testing for anti-PF4/H antibodies is performed only in patients with typical clinical symptoms of HIT improves diagnostic specificity of the laboratory assays without losing sensitivity, thereby helping to avoid overdiagnosis and resulting HIT overtreatment. Short-term re-exposure to heparin, especially given intraoperatively for cardiovascular surgery, is a reasonable therapeutic option in patients with a history of HIT who subsequently test negative for HIT antibodies. Organ failure(s), enhanced bleeding risks, and other characteristics require special considerations regarding non-heparin anticoagulation: Argatroban is the alternative anticoagulant with pharmacokinetics independent of renal function, but it has a prolonged half-life in case of impaired liver function. For bivalirudin, protocols during cardiopulmonary bypass surgery are established, and it is suitable for patients with liver insufficiency. A major issue of direct thrombin inhibitors are false high activated partial thromboplastin time values in patients with comorbidities affecting prothrombin, which can result in systematic underdosing of the drugs. This is not the case for danaparoid and fondaparinux, which can be monitored by anti-factor Xa assays, but have long half-lives and no suitable antidote. This review includes also information on management of on- and off-pump cardiac surgery, ventricular assist devices, percutaneous interventions, continuous renal replacement therapy, and extracorporeal membrane oxygenation in patients with HIT.

Laboratory Monitoring of Low-Molecular-Weight Heparin and Fondaparinux

Compared with older agents, low-molecular-weight heparins (LMWH) and fondaparinux offer improved bioavailability and more predictable, dose-independent clearance. While routine monitoring of coagulation parameters is not usually necessary with these agents, certain populations (including pregnant patients, children, obese patients, and patients with renal impairment) may benefit from the monitoring of anti-factor Xa activity, thromboelastography, or other coagulation assays to help guide therapy. The chromogenic anti-factor Xa assay is currently the gold standard for monitoring LMWH and fondaparinux therapy. Thromboelastography has been used to monitor LMWH therapy in special situations but is not needed for routine use.

Activated Partial Thromboplastin Time Monitoring of Unfractionated Heparin Therapy: Issues and Recommendations.

When administering unfractionated heparin (UFH), therapeutic levels of anticoagulation must be achieved rapidly and maintained consistently in the therapeutic range. The basic assays for monitoring UFH therapy are the activated partial thromboplastin time (APTT) and/or the chromogenic antifactor Xa or antithrombin assays. For many laboratories, the APTT is the preferred standard of practice; however, the APTT is a surrogate marker that only estimates the heparin concentration. Many factors, including patient variation, reagents of the APTT, UFH composition, and concentration can influence the APTT result. This article reviews various methods to determine the heparin therapeutic range and presents recommendations for the laboratory to establish an APTT heparin therapeutic range for all sizes of hospitals.

ヒーモスアイエル ヘパリン リキッド®を用いた抗Xa活性測定によるリバーロキサバン血中濃度モニタリングの臨床的意義

ヒーモスアイエル ヘパリン リキッド®を用いた抗Xa活性測定によるリバーロキサバン血中濃度モニタリングの臨床的意義

The Influence of Assay Selection on Prothrombin Time Measured in Patients Treated With Rivaroxaban for Nonvalvular Atrial Fibrillation.

Prothrombin time (PT) can provide a qualitative assessment of the relative intensity of anticoagulation by rivaroxaban. More than ten types of assay are available for the measurement of PT in clinical settings, but it is not yet fully understood whether their interactions with rivaroxaban are uniform or inconsistent. We examined 139 blood samples from patients taking rivaroxaban. We measured PT using five different commercially available assays. We also evaluated the estimated rivaroxaban concentration using a chromogenic anti-factor Xa assay. The median estimated concentration of rivaroxaban was 192 ng/ml (interquartile range 85-284 ng/ml). The correlation coefficient (r) between PT and the estimated concentrations of rivaroxaban was as follows: Thromborel S, r = 0.768; Thrombocheck PT, r = 0.861; Coagpia PT-N, r = 0.909; Neoplastin Plus, r = 0.882; and Triniclot PT Excel S, r = 0.870. The gradients of the regression plots differed more than fourfold, and the standard deviation of the regression line ranged from 1.001 to 2.980, which tended to be higher for the assays with the higher regression slope gradients. The estimated concentration of rivaroxaban varied greatly depending on the assay, so the PT measured in patients taking rivaroxaban should be interpreted with caution.

Evaluation of a Heparin-Calibrated Antifactor Xa Assay for Measuring the Anticoagulant Effect of Oral Direct Xa Inhibitors

The introduction of oral direct anti-Xa anticoagulants apixaban and rivaroxaban has significantly impacted the treatment and prevention of thromboembolic disease. Clinical scenarios exist in which a quantitative assessment for degree of anticoagulation due to these agents would aid management. The purpose of this work was to evaluate the chromogenic antifactor Xa assay calibrated with heparin standards at our institution for assessment of intensity of anticoagulation with rivaroxaban or apixaban in addition to its current use for unfractionated heparin or low-molecular-weight heparin. We also aimed to propose expected steady state peak and trough antifactor Xa activities for these agents based upon dosing regimens approved for nonvalvular atrial fibrillation. Antifactor Xa activity correlated very strongly with apixaban and rivaroxaban concentration in both spiked samples and treated patient plasma samples (r (2) = .99, P < .001). This correlation was observed over a broad range (20-500 ng/mL) of drug concentrations, as sample dilution with pooled normal plasma significantly extended the range of quantitative assessment. Based on drug concentrations previously published in pharmacokinetic studies, the expected steady state peak and trough antifactor Xa activity ranges for apixaban are 1.80 to 2.20 IU/mL and 0.70 to 1.10 IU/mL, respectively. For rivaroxaban, these ranges are 3.80 to 6.20 IU/mL and 0.60 to 1.00 IU/mL, respectively. In conclusion, our findings demonstrate that heparin-calibrated antifactor Xa activity correlates strongly with apixaban and rivaroxaban concentration. The dilution of samples allowed for this correlation to be extended over the majority of on-therapy drug concentrations.

New abstract created on Tuesday September 29, 2015 Immobilization of a novel anti-thrombotic drug on a metallic vascular stent

Event Abstract Back to Event New abstract created on Tuesday September 29, 2015 Immobilization of a novel anti-thrombotic drug on a metallic vascular stent Nicolas Bricout1, Jonathan Sobocinki1, 2, Adrien Hertault1, 2, William Laure3, Feng Chai1, Stéphan Haulon1, 2, Joel Lyskawa3 and Nicolas Blanchemain1 1 University of Lille, INSERM U1008 - Biomaterial Research Group, France 2 CHU de Lille, Aortic centre, Vascular Surgery, France 3 University of Lille, UMET, CNRS UMR 8207 Polymer Systems Engineering, France Introduction: In-stent restenosis (ISR) is promoted by platelet aggregation and thrombus formation occurring after stent implantation[1]. Drug-eluting stents have reduced the rate of ISR, but are associated with high risk of late acute stent thrombosis. Highly bioadhesive polydopamine (PDA) coating on Chromium-Cobalt (CrCo) substrate represents an interesting platform, which enables the immobilization of various relevant drugs promoting vascular wall healing[2] and may also directly improve endothelial cells proliferation[3]. This study aimed at developing and optimizing a PDA-coated CrCo stent functionalized with EP (224283, Endotis Pharma SA), a molecule that combines GPIIbIIIa antagonist and factor Xa inhibitor to reduce acute thrombosis and ISR[4]. Materials and Methods: CrCo substrates were coated by PDA as previously reported[3], and immersed in a polyethylenimine solution (PEI, pH8.5) overnight to react with PDA (by Michael addition and Schiff base reaction) in order to increase the amount of amine functions on the surface. This PDA-PEI coating enabled covalent immobilization of N-hydroxysuccinimide Biotin[5]. This key step permitted Avidin immobilization (via its high affinity for biotin), which can further interact with the biotin moiety of EP. The amount of amino groups formed on the PDA-coated surface before and after PEI addition was determined by orange acid II (OAII) colorimetric titration. Surface of samples was further characterized by Surface Plasmon Resonance (SPR) and Scanning Electron Microscopy (SEM). Cytocompatibility of functionalized surface was evaluated with human umbilical vein endothelial cells (HUVECs). The antithrombotic and antiplatelet activities of samples were evaluated by anti-factor Xa (anti-Xa) assay, lactate dehydrogenase (LDH) assay and SEM, respectively. Degradation kinetics of the EP-functionnalized samples in phosphate buffered saline (PBS) was studied under semi-dynamic condition and evaluated by monitoring anti-Xa activity. Results: OAII assay proved a significant increase in amino groups available on PDA-PEI surface (36.0 nM/cm2) compared to PDA ones (10.4 nM/cm2). The successive grafting of PEI, Biotin, and Avidin on PDA-coated samples was well evidenced by SPR (Figure 1) and SEM. Moreover, the immobilized EP on functionalized substrates exhibited a higher anti-Xa activity ( >1.81 U/mL) than that of control (0.09 U/mL), as depicted in Figure 2. These findings highlighted that EP was effectively grafted on the functionalized CrCo surface and preserved its biological activity. No alterations of HUVECs cell vitality were found on EP-functionalized samples. Degradation property of functionalized surface is under study. Conclusions: This study developed an innovative grafting process of an antithrombotic and antiplatelet molecule - EP(224283) on a PDA/PEI-coated CrCo via Biotin-Avidin interaction. This functionalized stent may offer a promising solution to reduce ISR and late acute thrombosis. AbbottVascular (Santa Clara, CA., USA) for providing vascular stents used for the purpose of the study; Endotis Pharma SA for generous supply of the EP224283; Alexandre Ung for his kind help in haematological analysis; French Society of Radiology (SFR) for providing financial support to this project

Fondaparinux monitoring in a patient with heparin-induced thrombocytopenia on hemodialysis

Heparin-induced thrombocytopenia associated to hemodialysis is rare. In case when citrate dialysis and/or non-heparin anticoagulants are not available, only possible medication to use for anticoagulation during hemodialysis is fondaparinux. However, laboratory monitoring of fondaparinux based on anti-Xa activity in dialysis patients has not been sufficiently documented yet. We created a local anti-factor Xa assay for measuring fondaparinux plasma concentration and efficacy in a patient with heparin-induced thrombocytopenia during hemodialysis. Fondaparinux given subcutaneously increases risk of adverse events due to its extended release and prolonged maintenance of toxic levels. When used intravenously fondaparinux remains safe, with reached steady-state level within dialysis and low risk of toxicity afterwards. Fondaparinux may be used as an alternative anticoagulant medication during hemodialysis in patients who develop heparin-induced thrombocytopenia. Adequate dose must be adjusted to patients’ dry weight (0.03 mg/kg intravenously) and fondaparinux anti-coagulation monitoring must be provided.