Activity Of Heparin Research Articles

Sample Dilution Is Not an Effective Mitigation for Hemolysis Interference in Anti-Xa Heparin Activity Assays

Abstract The presence of hemolysis or icterus can interfere with chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity), resulting in falsely lower estimates of heparin activity. Dilution is a common method for mitigating interference in spectroscopic assays that measure a single analyte by endpoint analysis and has been proposed as a solution for hemolysis interference in heparin activity assays. Heparin activity assays measure heparin/antithrombin complex activity using a chromogenic rate of absorbance change method. Heparin activity is a complex equilibrium of heparin binding to multiple proteins, including antithrombin, heparin cofactor II, platelet factor 4, histidine-rich glycoprotein, vitronectin, fibronectin, and others; some enhance while others neutralize heparin activity. The objective of this study was to determine how dilution affects heparin activity with different starting levels of antithrombin. First samples were diluted without interference (no hemolysis). Samples with 1 U/mL heparin in pooled normal plasma (PNP) and 50% PNP (to simulate 50% antithrombin levels) were diluted 1:1 with PNP versus OK buffer. Dilution resulted in nonlinear changes. Samples with 100% antithrombin recovered 88% of expected activity with PNP versus 74% with OK buffer; samples with 50% antithrombin recovered 111% with PNP versus 77% with OK buffer. Samples were then spiked with varying concentrations of hemolysate and 1:1 dilutions with PNP demonstrated nonlinear recoveries ranging from 41% to 84%. We were unable to recover the original heparin activity with or without hemolysis due to the complex interaction of initial heparin, antithrombin, and other binding protein concentrations with diluent antithrombin and other diluent heparin binding protein levels. When PNP is used as the diluent, the recovery will be lower when the initial antithrombin level is normal due to the addition of other heparin binding proteins; when the initial antithrombin level is low, the recovery is higher due to the addition of antithrombin from the diluent plasma. The recovery is always lower when buffer is used due to dilution of sample antithrombin. Careful consideration should be taken in evaluating the utility of sample dilution to mitigate interference from hemolysis and icterus in heparin activity assays, particularly in patients with fluctuating levels of antithrombin, such as those on extracorporeal life support or with liver failure.

Anti-inflammatory Surface Coatings Based on Polyelectrolyte Multilayers of Heparin and Polycationic Nanoparticles of Naproxen-Bearing Polymeric Drugs.

Immune response to biomaterials can produce chronic inflammation and fibrosis leading to implant failure, which is related to the surface properties of the biomaterials. This work describes the preparation and characterization of polyelectrolyte multilayer (PEM) coatings that combine the anti-inflammatory activity of heparin as polyanion with the potential release of Naproxen, a nonsteroidal anti-inflammatory drug from polymeric nanoparticles (NP) with cationic surface charge. The polyelectrolyte multilayers were characterized by physical methods to estimate multilayer growth, thickness, zeta potential, and topography. It was found that multilayers with NP had negative zeta potentials and expressed a viscoelastic behavior, while studies of topography showed that nanoparticles formed continuous surface coatings. THP-1-derived macrophages were used to study short-term anti-inflammatory activity (time scale 48 h), showing that PEM that contained heparin reduced cell adhesion and IL1-β secretion, when compared to those with polystyrenesulfonate, used as alternative polyanion in multilayer formation. On the other hand, the presence of NP in PEM was related to a reduced foreign body giant cell formation after 15 days, when compared to PEM that contained chitosan as alternative polycation, which suggests a long-term anti-inflammatory effect of Naproxen-containing nanoparticles. It was also shown that macrophages were able to take up NP from multilayers, which indicates a release of Naproxen by digestion of NP in the lysosomal compartment. These findings indicate that surface coatings composed of heparin and Naproxen-based NP on implants such as biosensors have the potential to attenuate foreign body reaction after implantation, which may improve the long-term functionality of implants.

Delivery of targeted gene therapies using a hybrid cryogel-coated prosthetic vascular graft.

ObjectivesThe success of prosthetic vascular grafts in the management of peripheral arterial disease is frequently limited by the development of anastomotic neointimal hyperplasia (ANIH), with the host response to prosthetic grafts beginning soon after implantation. To address this, we combine a platform of polyethylene terephthalate (PET) fabric with an applied cryogel layer containing biologic agents to create a bioactive prosthetic graft system, with the ability to deliver therapeutics targeting modulators of the ANIH-associated transcriptome response, along with antithrombotic agents.MethodsHybrid graft materials were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun (ePET), knitted PET (kPET), or woven PET (wPET). Arg-Gly-Asp (RGD) peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Physical properties such as swelling ratio, pore connectivity, shape recovery, and stiffness were evaluated. Human aortic endothelial cell (HAoEC) adherence was visualized using confocal microscopy after 24 hours and proliferation was evaluated with a resazurin-based assay for 7 days. Confocal microscopy was used to assess delivery of adeno-associated virus (AAV-GFP) after incubation of hybrid grafts with HAoECs. Heparin activity of the materials was measured using an anti-Xa assay.ResultsSEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. Hybrid materials showed a trend towards increased shape recovery, increased stiffness, decreased swelling ratio, and no difference in pore connectivity. HAoECs incorporated, adhered, and proliferated over 7 days on all materials. HAoECs were successfully transduced with AAV-GFP from the hybrid graft materials. Anti-Xa assay confirmed continued activity of heparin from all materials for over 7 days.ConclusionsWe have developed a bioactive prosthetic graft system with a cryogel coating capable of delivering biologic agents with antithrombotic activity. By applying the cryogel and selected agents onto PET prior to graft implantation, this study sets the stage for the system to be individualized and tailored to the patient, with bioengineering and targeted gene therapy strategies dovetailing to create an improved prosthetic graft adaptable to emerging knowledge and technologies.

Heparin and citrate additive carryover during blood collection.

Background Published evidence on the risk of additive carryover during phlebotomy remains elusive. We aimed to assess potential carryover of citrated and heparinized blood and the relative volume needed to bias clinical chemistry and coagulation tests. Methods We simulated standardized phlebotomies to quantify the risk of carryover of citrate and heparin additives in distilled water, using sodium and lithium as surrogates. We also investigated the effects of contamination of heparinized blood samples with increasing volumes of citrated blood and pure citrate on measurements of sodium, potassium, chloride, magnesium, total and ionized calcium and phosphate. Likewise, we studied the effects of contamination of citrated blood samples with increasing volumes of heparinized blood on heparin (anti-Xa) activity, lithium, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). We interpreted these results based on measurement deviations beyond analytical, biological and clinical significance. Results Standardized phlebotomy simulations revealed no significant differences in concentration of surrogate markers. Clinically significant alterations were observed after contamination of heparinized blood samples with volumes of citrated blood beyond 5-50 μL for ionized calcium and beyond 100-1000 μL for sodium, chloride and total calcium. Investigations of pure citrate carryover revealed similar results at somewhat lower volumes. Heparinized blood carryover showed clinically significant interference of coagulation testing at volumes beyond 5-100 μL. Conclusions Our results suggest that during a standardized phlebotomy, heparin or citrate contamination is highly unlikely. However, smaller volumes are sufficient to severely alter test results when deviating from phlebotomy guidelines.

Evaluation of the Activity of Heparin Injected into the Fully Implantable Catheter for Chemotherapy (Portocath) between Two Moments of Use

The aim of this study is to analyze whether heparin, used as a lock in fully implantable catheter for chemotherapy (portocath), maintains its activity even if it remains in the catheter for a long period of time. According to the institutional protocol, all catheters routinely use the lock solution with 3mL of heparinized solution after chemotherapy and the time interval between each change as lock in the catheters studied ranged from 7 to 30days. A total of 25 blood samples from 22 patients with 6 types of neoplasia on chemotherapy or not were collected according to routine, and the 10mL of liquid contained in the first aspirated reservoir/catheter (corresponding to the lock of the last section), were sent for laboratory analysis for prospectively studied with the following tests: anti-Xa, partially activated thromboplastin time (APTT), thrombin time (TT), reptilase, and thromboelastogram. Heparin activity was found in 96% of the anti-Xa and APTT tests. In relation to TT, 92% presented activity. The reptilase test was performed on 24 samples with significant time reduction in all of them. In the INTEM stage, the thromboelastometry test showed activity in 92% of samples and in the HEPTEM phase there was reduction in time in all samples. In all samples, the heparin activity was found to be independent of the time of use. We can conclude that lock of heparinized solution used in our service in fully implantable central venous catheters for chemotherapy was maintained with active heparin even after a long period of time (up to 30days), demonstrating that the half-life of the substance within the catheter is greater than its plasma half-life.

Cardiac Surgery without Donor Blood Transfusion at a Referral Cardiac Institute in Bangladesh

Background: Blood transfusion is always associated with some hazards despite economic and work burden on laboratory staffs. Pre-donation and blood conservation can help to avoid transfusion related hazards with good posto-perative outcome.
 Objective: The main objective of this study was to show the varieties of cardiac surgery without donor blood transfusion.
 Methodology: Patients were selected conveniently. Preoperative proper counseling was done to the guardians of minor and all the adult patients about the procedure and written informed consent was taken accordingly. All patients were operated under cardio-pulmonary bypass (CPB) following a standard protocol through mid-sternotomy, systemic heparinization, antegrade intermittent cold cardioplegic arrest of the heart. After weaning from CPB protamine was administered to neutralize the action of heparin. A pre-operative hemoglobin (Hb) and hematocrit (Hct) measured just before systemic heparinization. One bag of blood drawn from the patient’s body when the Hb and Hct more. Meticulous hemostasis done after weaning from cardio-pulmonary bypass. Both systemic and local tranexamic acid plus calcium injection was given before sternal wires are tighten. All the blood in the cardio-pulmonary bypass machine was returned at the end of operation. Patient’s hemoglobin and hematocrit checked to detect the need for transfusion. All other variables were studied.
 Results: Ten patients were operated without pre-operative blood transfusion of donor blood. Out of ten patients male=5 female=5, Male:Female=1:1 age range 5-26 years mean ±SD (13±-6.88) years. Preop diagnosis ASD 4/10=40%,VSD 3/10=30% TOF 2/10=20% AVR=1/10=10%. pre-operative Hb Range: 12.4-17.3 gm/dl. Mean ±SD (14.25±-1.66 gm/dl. Pre-operative Hct mean±SD (43.7±4.5). Per-operative Hct during CPB Mean±SD (24.9±4). Post-operative Hb mean ±SD (11.25gm±1.18gm). Post-operative Hct mean ±SD (34.3±3.80). Use of cell saver 1/8(12.5%) Post-operative blood loss for ASD,VSD, TOF and AVR were mean ±SD = 140±20.60ml, 156.44±33.84.320.80±60.22ml and 280 ml respectively. Varieties of ionotropes used were: Dopamine Dobutamin Adrenaline. Single ionotrope Dopa/Dobuta5/10=50% Double ionotrope Dopa/dobuta+Adrin=3/10(30%) tripple ionotrope 2/10(20%). Morbidity and mortality 0%. All the patients were discharged home uneventfully, in hospital outcome was good.
 Conclusion: In our setup we can do cardiac surgery without pre-operative donor blood use by following blood pre-donation and other blood conservative techniques.]
 Journal of National Institute of Neurosciences Bangladesh, 2019;5(1): 53-58

Detection and Extraction of Heparin from Camel Lungs.

Heparin is an essential drug used as an anticoagulant. Access to raw material suitable for heparin extraction is critical for creating a viable business opportunity. In Saudi Arabia, large amounts of raw material with potential for heparin extraction are wasted. To extract heparin and low-molecular-weight heparin (LMWH) from the camel lung, and measure its potency and activity. Heparin preparation included three steps: extraction, electrophoretic identification, and activity measurement. Fresh lung tissue (100 g) was minced and homogenized in a blender. Crude heparin extracts were prepared using Charles's or Volpi's method with slight modifications. Heparin was purified by electrophoresis using high-purity agarose gels in barium acetate buffer. The heparin activity of purified samples was assayed spectrophotometrically using commercial heparin kits. Charles's and Volpi's extraction methods were simple and easy to establish. The yield was 90 mg crude heparin per 100 g of camel lung tissue following Volpi's extraction protocol, whereas Charles's method did not yield any heparin. The separation of heparin and LMWH by gel electrophoresis resulted in sharp and clear product bands using material prepared according to Volpi's method. The heparin preparation had an anti-factor Xa activity of 37 IU/mg, indicating weak potency. Preparation of active heparin from camel lung tissue is a technology applicable in manufacturing. Further method development is needed to increase heparin purity and potency.

Cartridge-Based Thromboelastography Can Be Used to Monitor and Quantify the Activity of Unfractionated and Low-Molecular-Weight Heparins.

Thromboelastography is increasingly utilized in the management of bleeding and thrombotic complications where heparin management remains a cornerstone. This study assessed the feasibility of the cartridge-based TEG ® 6s system (Haemonetics Corp., Braintree, Massachusetts, United States) to monitor and quantify the effect of unfractionated and low-molecular-weight heparin (UFH and LMWH). Blood samples from healthy donors were spiked with UFH ( n = 23; 0–1.0 IU/mL) or LMWH (enoxaparin; n = 22; 0–1.5 IU/mL). Functional fibrinogen maximum amplitude (CFF.MA), RapidTEG activated clotting time (CRT.ACT), and kaolin and kaolin with heparinase reaction time (CK.R and CKH.R) were evaluated for their correlation with heparin concentrations, as well as the combination parameters ΔCK.R − CKH.R, ratio CK.R/CKH.R, and ratio CKH.R/CK.R. Nonlinear mixed-effect modelling was used to study the relationship between concentrations and parameters, and Bayesian classification modelling for the prediction of therapeutic ranges. CK.R and CRT.ACT strongly correlated with the activity of LMWH and UFH ( p < 0.001). Using combination parameters, heparin activity could be accurately quantified in the range of 0.05 to 0.8 IU/mL for UFH and 0.1 to 1.5 IU/mL for LMWH. CRT.ACT was able to quantify heparin activity at higher concentrations but was only different from the reference range ( p < 0.05) at >0.5 IU/mL for UFH and >1.5 IU/mL for LMWH. Combination parameters classified blood samples into subtherapeutic, therapeutic, and supratherapeutic heparin ranges, with an accuracy of >90% for UFH, and >78% for LMWH. This study suggests that TEG 6s can effectively monitor and quantify heparin activity for LMWH and UFH. Additionally, combination parameters can be used to classify blood samples into therapeutic ranges based on heparin activity.

Anticoagulant activity of porcine heparin: Structural-property relationship and semi-quantitative estimation by nuclear magnetic resonance (NMR) spectrometry

Heparin is a carbohydrate polymer, which is clinically used as an anticoagulant for the treatment of thrombotic disorders. The anticoagulant process is mainly mediated by the interaction of heparin with antithrombin followed by inhibition of clotting factors IIa (FIIa) and Xa (FXa). The influence of polymer disaccharide structure, average molecular weight and impurity profiling (e.g., chloride and water content) was investigated by NMR spectrometry and principal component analysis (PCA) for a representative dataset of porcine heparin samples (n = 509). A significant linear dependence was found between anticoagulant activity and scores on the third principal component (PC3) based on the non-targeted analysis of 1H NMR fingerprints. The correlation between average molecular values and anticoagulant activity for the 24 porcine heparin samples from two manufacturers was linear (R = 0.85) over typical values for porcine heparin preparations. Chloride and water contents were identified as negatively influencing factors for the actual activity values as their presence decrease the “pharmaceutically active” organic part of heparin preparations. Some suggestions regarding manufacturing process are made according to the results.

Lysine polymer neutralizes all types of heparin

The blood-thinning drug heparin halts the cascade of reactions that causes blood to clot. But too much heparin can lead to uncontrolled bleeding, so surgeons always keep protamine, the only US Food and Drug Administration–approved heparin-reversal agent, on hand. Chemists have now developed a promising substitute: a polymer that binds all three commonly used forms of heparin in plasma (ACS Med. Chem. Lett. 2019, DOI: 10.1021/acsmedchemlett.9b00090). Laurent Vial of the University of Lyon, Gerald Monard of the University of Lorraine, Jean-Christophe Gris of the University Hospital of Nimes, and colleagues made branched poly-l-lysine polymers of various molecular weights and tested their ability to stop heparin’s activity in plasma. One polymer almost entirely reversed the action of unfractionated heparin and outperformed protamine in tackling a low-molecular-weight form of heparin. It also stopped about 60% of the action of fondaparinux, a synthetic heparin, which is not neutralized by protamine. The polym...

Analysis of the Action of Quaternized Chitosans with Different Molecular Weight on Anticoagulant Activity of Heparins In Vitro.

Quaternized derivatives of chitosan with a substitution degree of 85-98% (highly substituted) synthesized from chitosans with a molecular weight of 5, 10, 20 kDa, with a degree of deacetylation of 89-98%, and the code numbers of QChit 5, QChit 10, QChit 20, respectively, completely neutralize antithrombin activity of unfractionated heparin and partially neutralize aXa activity of low-molecular-weight heparin (clexane), similar to protamine sulfate. The advantages of QChit 5 and QChit 10 over QChit 20 and protamine sulphate are the follows: the effect is achieved at lower concentrations and in greater concentration range; they do not promote platelet aggregation; in a concentration of 0.0072 mg/ml they do not destroy the erythrocyte membranes.

Interference in the anti-Xa heparin activity assay due to hemolysis and icterus during pediatric extracorporeal life support.

Chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity) are susceptible to interference from hemolysis and icterus. The purpose of this study was to better understand the effect of hemolysis and icterus on anti-Xa heparin activity and to predict the magnitude of the error. Increasing levels of hemoglobin and unconjugated bilirubin were added to pooled normal plasma or buffer containing known levels of heparin. Increased plasma hemoglobin or bilirubin produced falsely increased residual factor Xa activity as measured by the absorbance change (OD/min) in the Stago heparin activity assay. This increased absorbance change slope resulted in falsely lower estimates of heparin activity. The falsely lower heparin activity measurement occurred even when heparin was not present, indicating it was not due to heparin neutralization. In a sample containing 0.62±0.06 U/mL heparin and 228 mg/dL hemoglobin, the measured heparin activity was 0.41±0.03 U/mL, underestimating heparin activity by 0.21±0.07 U/mL. Interference occurred if plasma hemoglobin was above 70 mg/dL or bilirubin was above 16 mg/dL, which happened in 16%-26% of samples from pediatric patients on extracorporeal life support (ECLS). In conclusion, hemolysis and icterus were common in ECLS patients, leading to underestimates of unfractionated heparin activity and potentially higher doses of heparin than intended. The magnitude of the heparin activity measurement error could be predicted based on plasma hemoglobin and bilirubin levels until these levels exceeded the technical limits of the assay, ~230 mg/dL hemoglobin and 55 mg/dL bilirubin.

Dendrigraft of Poly-l-lysine as a Promising Candidate To Reverse Heparin-based Anticoagulants in Clinical Settings.

By using a combination of experimental and computational experiments, we demonstrated that a second-generation dendrigraft of poly-l-lysine neutralizes the anticoagulant activity of unfractionated heparin, low-molecular-weight heparin, and fondaparinux more efficiently than protamine does in human plasma, making this synthetic polymer a promising surrogate of this problematic protein in clinical settings.

The utilization of low molecular weight heparin-poloxamer associated Laponite nanoplatform for safe and efficient tumor therapy

In the present investigation, we have synthesized the dalteparin which is a kind of low molecular weight heparin (LMWH) and possess the antitumor and antiangiogenic efficacy with carboxylates poloxamer 407 to form a heparin-poloxamer nanogel (HP copolymer). The complex HP is capable of enhancing the efficacies, minimizing the side effects of dalteparin and exhibiting a good thermosensitivity. Therewith, the synthetic Laponite RDS (LR) nanosilicate embarked with doxorubicin (DOX) at a satisfactory high drug entrapment efficiency was integrated with the complex HP and thus constituted a newfangled biocompatible injectable temperature-sensitive hydrogel. To our delight, consociation with 2.5 w/v % of LR nanodisks, HP at the concentration of 5 w/v % was sufficient to execute the solution-gel transition at animal heat, while 17.5 w/v % of P-407 was needed for fabricating the LR-P hydrogel. Simultaneously, LR-HP possesses a more preferable syringeability. Furthermore, the release behavior in vitro for the DOX@LR-HP demonstrated as extended and controlled manner, wherein, the drug-eluting profile was regulated by the LR nanocomposites. Moreover, based on synergic action of heparin and drug, DOX@LR-HP hydrogels demonstrated the best antitumor efficacy in vitro and in vivo. What is noteworthy is that through the course of treatment, the reflected anticancer efficacy direct at the established xenograft S180 sarcoma tumor was provided by the single administration of the DOX@LR-HP hybrid gel. This excellent long-term sustainable-anticancer function in vivo demonstrating the prospect of this nanohybrid-gel combination as an estimable focal drug delivery vehicle for treatment of cancer.

Chronic Ovine Studies Demonstrate Low Thromboembolic Risk in the Penn State Infant Ventricular Assist Device.

Mechanical circulatory support for children under 6 years of age remains a challenge. This article describes the preclinical status and the results of recent animal testing with the Penn State Infant Left Ventricular Assist Device (VAD). The objectives have been to 1) demonstrate acceptably low thromboembolic risk to support Food and Drug Administration approval, 2) challenge the device by using minimal to no anticoagulation in order to identify any design or manufacturing weaknesses, and 3) improve our understanding of device thrombogenicity in the ovine animal model, using multicomponent measurements of the coagulation system and renal ischemia quantification, in order to better correlate animal results with human results.The Infant VAD was implanted as a left VAD (LVAD) in 18-29 kg lambs. Twelve LVAD and five surgical sham animals were electively terminated after approximately 30 or 60 days. Anticoagulation was by unfractionated heparin targeting thromboelastography R times of 2x normal (n = 6) or 1x normal (n = 6) resulting in negligible heparin activity as measured by anti-Xa assay (<0.1 IU/ml). Platelet inhibitors were not used.There were no clinically evident strokes or evidence of end organ dysfunction in any of the 12 electively terminated LVAD studies. The degree of renal ischemic lesions in device animals was not significantly different than that found in five surgical sham studies, demonstrating minimal device thromboembolism.In summary, these results in a challenging animal test protocol support the conclusion that the Penn State Infant VAD has a low thromboembolic risk and may allow lower levels of anticoagulation.

Anti-Factor Xa–Based Monitoring of Unfractionated Heparin: Clinical Outcomes in a Pediatric Cohort

To assess clinical outcomes in children treated with unfractionated heparin and monitored using an anti-factor Xa (Anti-FXa)-based nomogram. We also sought to assess the correlation between activated partial thromboplastin time (APTT) and Anti-FXa. This was a single-center, observational cohort study conducted over a 20-month period that included all pediatric patients (<21years) who received therapeutic unfractionated heparin and were monitored using an anti-FXa-based nomogram. In total, 95 patients met prespecified inclusion criteria, and 1098 pairs of APTT and Anti-FXa measurements were performed. The median unfractionated heparin dose required to reach therapeutic Anti-FXa goal was significantly greater in infants compared with older children (P<.0001). The median time to achieve therapeutic Anti-FXa was 10hours (range 2-96hours) and was significantly shorter in patients who received a bolus compared with those who did not (P = .03). Five (5.3%) major bleeding events were noted. Age, peak Anti-FXa, peak APTT, lowest platelet count, and fibrinogen were not predictive of major and clinically relevant nonmajor bleeds. Moderate correlation between the APTT and Anti-FXa (r=0.75; 95% CI 0.72-0.77) assays was appreciated. Using an anti-FXa-based nomogram to monitor unfractionated heparin in children is feasible. Although moderate correlation was observed between the APTT and Anti-FXa assays, the APTT frequently overestimated heparin activity. Safety and efficacy of an Anti-FXa nomogram needs further validation.

Heparin prevents oxidative stress-induced apoptosis in human decidualized endometrial stromal cells.

Clinical trials have shown that administering heparin during the luteal phase has beneficial effects on implantation and live birth rates. Heparin exerts direct effects on decidual human endometrial stromal cells (HESCs), which are independent of its anticoagulant effect. However, the accurate effects of heparin on the decidualization process remain unidentified. Here, we demonstrate that HESCs become dramatically resistant to oxidative stress upon decidualization, and we hypothesize a possible direct action of heparin on the decidualization of HESCs, which would lead to improved implantation. To test this hypothesis, we established primary HESC cultures and propagated them, and then we decidualized confluent cultures with 8-bromo-cAMP, with medroxyprogesterone acetate, and with or without heparin. We treated the cells with hydrogen peroxide (H2O2) as a source of reactive oxygen species (ROS). Adding heparin to decidualized HESCs induced prolactin secretion. Decidualized HESCs treated with heparin were prevented from undergoing apoptosis induced by oxidative stress. Heparin induced nuclear accumulation of the forkhead transcription factor FOXO1 and expression of its downstream target, the ROS scavenger superoxide dismutase 2. These results demonstrate that heparin-treated decidualized HESCs acquired further resistance to oxidative stress, suggesting that heparin may improve the implantation environment.

DISORDERS OF COAGULATION STATUS AND HAEMOSTASIS AS PROGNOSTIC PARAMETERS OF IMMEDIATE AND EARLY RESULTS AFTER SURGICAL MYOCARDIAL REVASCULARISATION

Surgical myocardial revascularization is one of the most commonly performed surgical procedures in the world. Over time, with the development of technology and modern diagnostic procedures, as well as the advancement of surgical techniques, the mortality rate for elective uncomplicated cases has fallen to below 2%. Nevertheless, despite the exceptional development of the surgical techiniques, the rate of postoperative complications, that can compromise the patients, is over 10%. The aim of this study was to define a group of patients with an increased risk of postoperative complications depending on the disorders of coagulation status and haemostasis. Twenty eight patients who underwent surgical revascularization of the myocardium were included in this prospective, non-randomized study. The study was conducted at the Clinic for Cardiac Surgery, Clinical Center Nis, from January to April 2017. Preoperatively as well as 3 hours, 24 hours, 48 hours, 3 days, and 5 days postoperatively, the following parameters were determined: blood count, inflammation parameters (C reactive protein, presepsin); coagulation status (prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (APTT), fibrinogen, anti-thrombin III, D dimer). The only preoperative independent prognostic parameter for increased postoperative drainage was INR. Activated clothing time (ACT) was an independent postoperative prognostic parameter of increased postoperative drainage, probably due to delayed or prolonged heparin activity. Inflammation parameters showed no association with the onset of postoperative complications. In relation to patients without bleeding, in patients with bleeding, significantly higher values of urea and the difference in APTT values, preoperatively and at the end of the monitoring period, were detected. Multivariate logistic regression analysis, confirmed the difference in APTT values preoperatively and at the end of the monitoring period, as the only factor, significantly associated with the risk of bleeding. Multivariate linear regression analysis, confirmed the value of the urea, as the only factor significantly associated with the change in total allogeneic transfusion value. Increase in urea levels is associated with an increase in the total amount of allogeneic transfusion. Correlation analysis showed that the increased number of days in the intensive care was significantly associated with female gender, number of grafts, prolonged ECC, clamping time, hematocrit (HCT), PT, INR preoperatively and at the end of the follow-up period. Surgical myocardial revascularization is a safe method with a minimal morbidity rate. Using of modern methods for the preoperative monitoring of haemostasis may significantly reduce the risk of postoperative bleeding and the need for transfusion of red blood cells and other blood derivatives.

Bivalirudin Anticoagulation for an Infant with Hyperbilirubinemia and Elevated Plasma-Free Hemoglobin on ECMO

Heparin has been used for decades as an anticoagulant in patients on mechanical circulatory support, which includes extracorporeal membrane oxygenation (ECMO) and ventricular assist devices. Bivalirudin is a direct thrombin inhibitor that can be used as an alternative anticoagulant in neonates and infants demonstrating inaccurate heparin monitoring. We report a case of a 2-month-old male child who was placed on ECMO for severe acute respiratory distress syndrome. His ECMO course was complicated by severe hemolysis and hyperbilirubinemia, which precluded accurate monitoring of heparin activity. Bivalirudin was successfully used for anticoagulation in this patient.

Monitoring of heparins in antithrombin-deficient patients

IntroductionHeparins exert their anticoagulant effect through activation of antithrombin. Whether antithrombin deficiency leads to clinically relevantly reduced anti-Xa activity of heparins is unknown. We investigated the relation between antithrombin deficiency and anti-Xa activity measurements of plasma samples spiked with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). Materials and methodsPlasma samples from 34 antithrombin-deficient subjects and 17 family controls were spiked with UFH and LMWH (nadroparin) aimed to correspond with an anti-Xa activity of 0.8 IU/mL. Antithrombin, β-antithrombin and anti-Xa activities were measured. ResultsMean anti-Xa activity with LWMH was 0.55 IU/mL (0.30–0.74) (recovery 69%, 38–93%) in antithrombin-deficient subjects and 0.82 (0.71–0.89) IU/mL in controls (recovery 103%, 89–111%). Expected anti-Xa measurements after LMWH spiking were found in 17/17 non-deficient subjects and in 8/34 antithrombin-deficient subjects. Anti-Xa measurements in the expected range (0.6–1.0 IU/mL) after UFH spiking were found in 17/17 non-deficient subjects and in 1/22 antithrombin-deficient subjects. Antithrombin activity correlated with anti-Xa activity of UFH (R = 0.77) and LMWH (R = 0.66). Mixing studies of pooled normal plasma and antithrombin-deficient plasma showed that anti-Xa recovery was linearly reduced with antithrombin activity decreasing below 100%. ConclusionsReduced antithrombin activity causes significantly reduced anti-Xa levels. Standard LWMH- or UFH-doses are likely to lead to under treatment in antithrombin-deficient individuals.