Heparin anti-Xa Activity Research Articles

Pleiotropic Effects of Heparin and its Monitoring in the Clinical Practice.

Unfractionated heparin (UFH) was uncovered in 1916, has been used as an anticoagulant since 1935, and has been listed in the World Health Organization's Model List of Essential Medicines. Despite the availability of many other anticoagulants, the use of heparin (either low molecular weight heparin [LMWH] or UFH) is still substantial. Heparin has pleotropic effects including anticoagulant and several nonanticoagulant properties such as antiproliferative, anti-inflammatory activity, and anticomplement effects. Although UFH has been widely replaced by LMWH, UFH is still the preferred anticoagulant of choice for patients undergoing cardiopulmonary bypass surgery, extracorporeal membrane oxygenation, and patients with high-risk mechanical cardiac valves requiring temporary bridging with a parenteral anticoagulant. UFH is a highly negatively charged molecule and binds many positively charged molecules, hence has unpredictable pharmacokinetics, and variable anticoagulant effect on an individual patient basis. Therefore, anticoagulant effects of UFH may not be proportional to the dose of UFH given to any individual patient. In this review, we discuss the anticoagulant and nonanticoagulant activities of UFH, differences between UFH and LMWH, when to use UFH, different methods of monitoring the anticoagulant effects of UFH (including activated partial thromboplastin time, heparin anti-Xa activity level, and activated clotting time), while discussing pros and cons related to each method and comparison of clinical outcomes in patients treated with UFH monitored with different methods based on available evidence.

Development of a Rapid Qualitative Screen for Anticoagulant Presence.

Direct oral anticoagulants (DOACs) and fondaparinux with stable pharmacokinetics are commonly used anticoagulants for outpatient care. Due to the lack of monitoring requirements, drug-specific assays are not available in most hospital laboratories, but drug levels are needed in some urgent/emergency situations. This study describes the development of a qualitative screen for the presence of DOAC or fondaparinux using coagulation tests found in most laboratories. The DOAC screen is composed of a heparin anti-Xa activity assay and thrombin time (TT) assay. The STA®-Liquid-Anti-Xa assay calibrated with Stago Multi Hep® and STA®-TT were run on STA-R Max® analyzers. The anti-Xa activity and TT assays were repeated 5 times in samples of commercially available calibrators and controls for each drug: fondaparinux, dabigatran, rivaroxaban, apixaban, and edoxaban. Statistical analysis and correlations were performed for anti-Xa activity and TT results for each drug and pooled normal plasma. A significant correlation was found between heparin-calibrated anti-Xa levels and fondaparinux, rivaroxaban, apixiban, and edoxaban (r2 = 0.99-1.0). Dabigatran showed a strong linear correlation (r2 = 0.99) with TT. Anti-Xa levels >0.3 IU/mL and TT >25 seconds were determined as cutoffs at our lab for the detection of clinically relevant drug levels of factor Xa inhibitor and direct thrombin inhibitor, respectively. Our study demonstrates that commonly available heparin anti-Xa activity and TT assays can be used to qualitatively detect DOACs and fondaparinux and provides a method to establish a qualitative interpretation.

Usefullness of Heparin Calibrated Anti-Xa Activity to Assess Anticoagulant Activity of Apixaban and Rivaroxaban in Emergency Patients Scheduled for Acute Interventions.

(1) Background: Direct oral anticoagulants (DOACs) require monitoring in some critical clinical situations. The specific tests for DOAC monitoring are not yet available in all labs. The aim of this study was to evaluate if a unique, more widespread heparin-calibrated anti-Xa assay could be suitable to estimate the concentrations of apixaban and rivaroxaban in order to establish an algorithm helping our clinicians in their therapeutic decision for patients treated with DOACs in emergencies. (2) Methods: A first retrospective part allowed us to determine of a conversion factor between the measured DOAC concentration and the deducted anti-Xa heparin activity based on optic density. During the second prospective part, both DOAC concentration (ng/mL) and anti-Xa activity heparin (UI/mL) were measured on the same sample, and the previously determined conversion factor was applied to each UI/mL value. We then compared the calculated and measured DOAC concentration values. (3) Results: The analysis of the derivation cohort confirmed a good correlation, especially between the anti-Xa heparin activity and the apixaban concentrations (r = 0.97). Additionally, we determined heparin-calibrated anti-Xa assay cut-offs for invasive procedures at 0.3 UI/mL and for intravenous thrombolysis at 0.51 UI/mL using ROC curves with a sensitivity at 98% and specificity at 95% for 0.3 UI/mL and a sensitivity at 97.7% and specificity at 88.2% for the cut-off of 0.51 UI/mL. In the validation cohort, we confirmed the agreement between measured and calculated DOAC concentrations for the low values, especially around cut-offs with an excellent negative predictive value for 0.51 UI/mL (94% for apixaban and 100% for rivaroxaban) and a good negative predictive value for 0.3 UI/mL (83.3% for apixaban and 85.7% for rivaroxaban). (4) Conclusions: Our results confirm that it is possible to correctly predict or exclude the presence of apixaban/rivaroxaban in emergency situations when specific tests are not readily available.

Correlation between heparin anti-Xa activity and thromboelastography in adult critically ill COVID-19 patients.

Severe coronavirus disease 2019 (COVID-19) increases the risk of thrombotic complications with unfractionated heparin (UFH) as a commonly used agent in managing venous thromboembolism (VTE). The optimal anticoagulation intensity and monitoring parameters in intensive care unit (ICU) COVID-19 patients remains controversial. The primary study aim was to evaluate the relationship between anti-Xa and thromboelastography (TEG) reaction (R) time in patients with severe COVID-19 receiving therapeutic UFH infusions. Single-center, retrospective study conducted over a 15-month period (2020-2021). Academic medical center (Banner University Medical Center Phoenix). Adult patients with severe COVID-19 administered therapeutic UFH infusions with one or more corresponding TEG, and anti-Xa assessments drawn within ≤2 hours of each other were included. The primary end point was the correlation between anti-Xa and TEG R time. Secondary aims were to describe the correlation between activated partial thromboplastin time (aPTT) and TEG R time, as well as clinical outcomes. Pearson's coefficient was used to evaluate the correlation using a kappa measure of agreement.

Conversion From Activated Clotting Time to Anti-Xa Heparin Activity Assay for Heparin Monitoring During Extracorporeal Membrane Oxygenation.

Anticoagulation with unfractionated heparin remains the most common therapy used to prevent circuit thrombosis during extracorporeal membrane oxygenation, but no consensus exists on the optimal method or targets for heparin monitoring. From 2015 to 2018, we switched from monitoring heparin during extracorporeal membrane oxygenation using activated clotting times to anti-Xa heparin activity assays. This study describes the transition from activated clotting time to anti-Xa heparin activity assay monitoring and the associated clinical changes. Retrospective analysis at single institution. Referral Children's Hospital. A total of 145 pediatric patients over 152 extracorporeal membrane oxygenation runs using 206 extracorporeal membrane oxygenation circuits. Anticoagulation protocol quality improvement. From 2015 to 2018, heparin monitoring during extracorporeal membrane oxygenation changed from hourly activated clotting time to anti-Xa heparin activity assay every 6 hours with an associated 75% reduction in the circuit changes per extracorporeal membrane oxygenation day. Over the 4 years, patients with an average anti-Xa heparin activity assay of at least 0.25 U/mL showed a 59% reduction in circuit changes per extracorporeal membrane oxygenation day compared with less than 0.15 U/mL. In addition to its association with reduced circuit changes, anti-Xa heparin activity assay monitoring was also associated with reduced heparin dose changes per day from 11 ± 4 to 2 ± 1 (p < 0.001), smaller heparin dose changes (less variation in dose), and reduced diagnostic phlebotomy volumes from 41 ± 6 to 25 ± 11 mL/day (p < 0.001). The number of patients with reported bleeding decreased from 69% using activated clotting time to 51% (p = 0.03). Transfusion rates did not change. Over 4 years, we replaced the activated clotting time assay with the anti-Xa heparin activity assay for heparin monitoring during extracorporeal membrane oxygenation. Minimum anti-Xa heparin activity assay levels of 0.25 U/mL were associated with reduced circuit changes. Further studies are needed to determine the optimum anti-Xa heparin activity assay therapeutic range during extracorporeal membrane oxygenation.

Impact of exogenous antithrombin on low molecular weight heparin anti-Xa activity assays in a pediatric and young adult leukemia and lymphoma cohort with variable antithrombin levels.

Low molecular weight heparin (LMWH) remains the most commonly prescribed pediatric anticoagulant. There is debate whether LMWH anti-Xa assays with or without exogenous antithrombin (AT) best reflect anticoagulation effect, and how much discrepancy exists between assay types. We assessed the effect of variable AT activity on LMWH anti-Xa levels in plasma samples from anticoagulated pediatric and young adult acute lymphoblastic leukemia and lymphoma (ALL/L) patients, using two instruments and their commercial kits with and without exogenous AT (ie, four platforms). We analyzed LMWH anti-Xa levels on 60 plasma samples with known AT activity from 12 enoxaparin-treated ALL/L patients, using four commercial kits from Siemens and Stago containing AT or not, on Siemens BCS and Stago STA R Max, respectively. Of 236/240 samples with interpretable results, mean AT activity was 80% (46-138%). Correlation was acceptable for published kit ranges of LMWH anti-Xa levels when comparing kits containing AT (r=0.82, P<.0001), or not (r=0.93, P<.0001), and within a manufacturer (Berichrom to Innovance, r=0.92, P<.0001; Stachrom to STA-Liquid Anti-Xa r=0.98, P<.0001). LMWH anti-Xa levels were lower in platforms without added AT (P<.0001). For Stago kits, this effect increased when AT<70% (P=.001, n=19, mean 56%). Assay variability, measured as mean percent difference, was less pronounced with Stago kits (14.7%, n=49) than Siemens (41.9%, n=50). Although LMWH levels from anti-Xa assays with added AT trend higher than in those without, correlation was fairly good between platforms in pediatric ALL/L plasmas, even when AT activity was <70%.

Heparin Anti-Xa Activity, a Readily Available Unique Test to Quantify Apixaban, Rivaroxaban, Fondaparinux, and Danaparoid Levels.

Despite their usefulness in perioperative and acute care settings, factor-Xa inhibitor-specific assays are scarcely available, contrary to heparin anti-Xa assay. We assessed whether the heparin anti-Xa assay can (1) be used as a screening test to rule out apixaban, rivaroxaban, fondaparinux, and danaparoid levels that contraindicate invasive procedures according to current guidelines (>30 ng·mL-1, >30 ng·mL-1, >0.1 µg·mL-1, and >0.1 IU·mL-1, respectively), (2) quantify the anticoagulant level if found significant, that is, if it exceeded the abovementioned threshold. In the derivation cohort then in the validation cohort, via receiver operating characteristics (ROC) curve analysis, we evaluated the ability of heparin anti-Xa assay to detect levels of factor-Xa inhibitors above or below the abovementioned safety thresholds recommended for an invasive procedure (screening test). Among samples with relevant levels of factor-Xa inhibitor, we determined the conversion factor linking the measured level and heparin anti-Xa activity in a derivation cohort. In a validation cohort, the estimated level of each factor-Xa inhibitor was thus inferred from heparin anti-Xa activity. The agreement between measured and estimated levels of factor-Xa inhibitors was assessed. Among 989 (355 patients) and 756 blood samples (420 patients) in the derivation and validation cohort, there was a strong linear relationship between heparin anti-Xa activities and factor-Xa inhibitors measured level (r = 0.99 [95% confidence interval {CI}, 0.99-0.99]). In the derivation cohort, heparin anti-Xa activity ≤0.2, ≤0.3, <0.1, <0.1 IU·mL-1 reliably ruled out a relevant level of apixaban, rivaroxaban, fondaparinux, and danaparoid, respectively (area under the ROC curve ≥0.99). In the validation cohort, these cutoffs yielded excellent classification accuracy (≥96%). If this screening test indicated relevant level of factor-Xa inhibitor, estimated and measured levels closely agreed (Lin's correlation coefficient close to its maximal value: 95% CI, 0.99-0.99). More than 96% of the estimated levels fell into the predefined range of acceptability (ie, 80%-120% of the measured level). A unique simple test already widely used to assay heparin was also useful for quantifying these 4 other anticoagulants. Both clinical and economic impacts of these findings should be assessed in a specific study.

Transitioning hospitalized patients from rivaroxaban or apixaban to a continuous unfractionated heparin infusion: A retrospective review.

To determine a patient's clinical course based on the use of an activated partial thromboplastin time (aPTT) or heparin anti-Xa assay when transitioning from rivaroxaban or apixaban to an unfractionated heparin infusion. A retrospective chart review was conducted to investigate how unfractionated heparin infusions were managed at a tertiary care hospital in the setting of recent apixaban or rivaroxaban administration. Patients were separated into 2 cohorts based on the chosen heparin infusion monitoring assay: heparin anti-Xa or aPTT. The primary composite outcome was total number of bleeding and thrombotic events; the secondary composite outcome was average incidence of heparin infusion holds and rate changes per patient. Data were collected from 76 patients (heparin anti-Xa = 69, aPTT = 7). Due to the limited number of patients within the aPTT cohort, this data was excluded from the analysis, and heparin anti-Xa descriptive statistics were reported without statistical comparisons. In the heparin anti-Xa group, a total of 10 bleeds and 1 thrombus were discovered. Additionally, the average number of infusion holds and rate changes was 0.841 and 2.65 times per patient, respectively, for those patients monitored via heparin anti-Xa assay. In the presence of a recently administered oral anti-Xa anticoagulant, more down-titrations occurred in the initial 6 hours of the heparin infusion when measuring anti-Xa activity, and most up-titrations occurred after 36 hours. Baseline heparin anti-Xa activity may be a useful tool to identify patients with residual plasma concentrations of apixaban and rivaroxaban to help better individualize heparin therapy.

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.

Discordant Partial Thromboplastin Time (PTT) vs Anti-Xa Heparin Activity.

To determine the relationship between baseline variations in the partial thromboplastin time (PTT) and the discordance between the PTT and anti-Xa heparin activity (anti-Xa) during heparin therapy. The baseline PTT on heparin was determined using automated heparin neutralization with protamine (prPTT). The prPTT was used to calculate a baseline-corrected PTT on heparin to reduce discordance with anti-Xa measurements. The prPTT removed up to 1 U/mL of heparin, returning baseline values for normal, factor-deficient, and lupus inhibitor plasmas. A prolonged prPTT was seen in 97 (53%) of 182 samples from heparinized patients. The heparinized PTT was discordant compared with anti-Xa in 64 (35%) of 182 samples and 43 (67%) of 64 discordant samples, and 46% of concordant samples showed a prolonged prPTT. A baseline-corrected PTT reduced discordance with anti-Xa measurements by 64%. PTT/anti-Xa discordance due to baseline PTT prolongation could be reduced using a baseline-corrected PTT.

Evaluation of Unfractionated Heparin Dosing for Thromboprophylaxis using Anti-Xa Levels in Obese Critically Ill Patients: A Retrospective Study

Purpose: This study aims to evaluate whether current dosing recommendations of UFH for the prevention of thromboembolism is appropriate in obese critically ill patients utilizing Anti-Xa levels. Materials and methods: A pilot, observational and retrospective chart review of critically ill patients was evaluated. Patients aged ≥ 18 years were included if they received subcutaneous UFH for thromboprophylaxis and had a recorded Anti-Xa level taken four to five h after the administration of UFH. Identified patients were categorized into two groups: BMI<30 kg/m2 or BMI ≥ 30 kg/m2. Anti-Xa heparin activity levels were then compared. Statistical analysis was performed using Student’s t-test. Results: A total of 130 adult ICU patients were included in this study. For all critically ill patients receiving 5,000 units of subcutaneous heparin every 8 h, there was no statistically significant difference in Anti-Xa levels between the groups (p=0.287). There was no incidence of DVT/ PE reported from either group. Conclusion: This study suggests that there is no significant difference in Anti- Xa levels between obese critically ill patients and non-obese patients receiving traditional UFH dosing for thromboprophylaxis.

Are all heparins safe for on-pump heart surgery?

ABSTRACTBackground: Intravenous Panpharma heparin® was used in all on-pump cardiac surgery in our heart-surgery department for a short period. This brand of heparin replaced the previous Choay heparin® heparin supplied by the Sanofi-Aventis Laboratory. Unusual postoperative bleedings over this period prompted us to evaluate postoperative hemostasis by comparing these two heparins.Methods: We compared data from patients who had undergone on-pump cardiac surgery during Panpharma heparin® period (group P, 257 patients) to those how received Choay heparin® (group C, 194 patients).Results: Despite group P receiving a significantly lower dose of heparin (mean dose 21,000 IU/CEC) compared to group C (mean dose 22,000 IU/CEC) (p = 0.05), the number of surgical re-explorations needed to perfect postoperative hemostasis was significantly higher for group P (3.5% vs. 0) (p = 0.01). Heparin anti-Xa activity after surgery was higher in group P at postoperative h1 and h12 compared to group C, which explained reoperations for hemostasis.Conclusion: Despite standardization, variations remain regarding anticoagulant activity between different manufacturing processes and heparin preparations. Surgical teams need to be aware that the biological effects of different brands of heparin may not be as expected and could endanger a usually safe procedure, such as cardiac surgery.

Assessment of anti-factor Xa activity of heparin in binary parenteral nutrition admixtures for premature neonates

An in vitro study was carried out to determine the anti-Xa activity of heparin in binary parenteral nutrition (BPN) admixtures for premature neonates in our neonatal intensive care unit (NICU) after a 24-hour infusion, as well as to assess drug interaction with a 50% glucose solution. Two types of bags were prepared: (1) BPN admixtures (composition defined in the NICU) including sodium heparin at 77 UI/mL and (2) bags containing only G50% with sodium heparin at 193 UI/mL. The anti-Xa activity of heparin was measured in bags at T0, after the 24-hour infusion and in eluates at the outlet of the infusion line after 24hours, using a validated chromogenic anti-Xa method. Comparisons of the mean concentration observed with the theoretical value for anti-Xa activity were performed with the Student t-test. Mean values of anti-Xa activity do not differ significantly from the values expected for all conditions. We found a slight variation in anti-Xa activity when infused over 24hours for both types of bags, with and without in-line filtration, showing that heparin remains stable during this infusion period in both BPN admixtures and G50%.

The myth of incompatibility of gentamicin and heparin revisited

Earlier reports indicated that gentamicin should not be mixed with heparin due to precipitation and drug mixture incompatibility. With the recent increased interest in antibiotic lock technique for prophylaxis or adjuvant therapy for hemodialysis catheter-associated infections, it became prudent to reexamine the issue. A mixture of gentamicin (100 µg/mL) and heparin (5,000 U/mL) in normal saline was stored for up to 4 weeks at 4ºC. Gentamicin concentration and bactericidal and anticoagulant activities were measured for up to 4 weeks of storage. The mixture did not show any turbidity or precipitation after fresh preparation, at 48 hours or 4 weeks of storage at 4ºC. Gentamicin concentration (mean ± SD) was 89 ± 7.5 µg/mL, 87 ± 1.0 µg/mL and 85 ± 3.0 µg/mL; gentamicin bactericidal activity was at 1:256 ± 0, 1:256 ± 0 and 1:213 ± 74 dilutions, after fresh preparation, at 48 hours or 4 weeks of storage at 4ºC, respectively (p=NS). Heparin anti-Xa activity was 7,500 ± 990 U/mL and 6,700 ± 707 U/mL, fresh mixture vs. after 4 weeks of storage at 4ºC, respectively (p=NS). In our in vitro experiments we showed that a mixture of gentamicin (100 µg/mL) and heparin (5,000 U/mL) in normal saline retained its gentamicin concentration, bactericidal activity and anticoagulant activity for up to 4 weeks of storage at 4ºC. We also present a brief review of the literature on this subject.

Heparin anticoagulation in patients undergoing off-pump and on-pump coronary bypass surgery

The authors analyzed the coagulation data of patients who underwent on-pump coronary artery bypass graft (CABG) or off-pump coronary artery bypass surgery (OPCAB) in a randomized prospective trial. CABG and OPCAB patients received heparin anticoagulation at 400 U x kg(-1), and 180 U x kg(-1) plus 3000 U every 30 min, respectively. In addition, OPCAB patients received a rectal aspirin, 650 mg, during the procedure. Perioperative coagulation test results (platelet count, fibrinogen, prothrombin time, partial thromboplastin time [PTT], activated clotting time [ACT], and thromboelastography [TEG; Haemoscope] were collected from CABG (n = 99) and OPCAB (n = 98) patients. Residual heparin activity after protamine was measured, using an anti-activated factor X (Xa) assay, in 10 patients from each group. Our study showed that the current anticoagulation regimen in the OPCAB patients achieved a peak ACT of 445 +/- 73 s, and it preserved platelet counts and fibrinogen levels. A residual heparin effect was detected, with residual anti-Xa heparin activity of 0.2 U x ml(-1) up to 2 h after surgery in the OPCAB group. Despite the residual anticoagulation, the OPCAB group had a similar TEG index of native blood, postoperative chest tube drainage, and non-erythrocyte transfusion rate as compared with the CABG group. We have shown that the heparin anticoagulation regimen in OPCAB patients does not lead to an immediate hypercoagulable state. Total doses of heparin and protamine were lower in the OPCAB group compared with the CABG group, and there was a residual heparin effect on TEG and PTT in the early postoperative period in the OPCAB group.

An Evaluation of the Effects of a Standard Heparin Dose on Thrombin Inhibition During Cardiopulmonary Bypass in Neonates

We compared the adequacy of heparinization in neonates and older children undergoing cardiopulmonary bypass (CPB) by measuring heparin activity, thrombin formation, and thrombin activity. Ten neonates and 10 older children were administered 400 U/kg of heparin before CPB. Heparin anti-Xa activity, prothrombin fragment 1.2 (F1.2), and fibrinopeptide A (FPA) were measured at baseline, after 30 min on CPB, immediately post-CPB, and 3 and 24 h post-CPB. Heparin anti-Xa activity was significantly decreased during and immediately post-CPB in the neonatal group. F1.2 and FPA levels in neonates were significantly higher at baseline, decreased with the commencement of CPB, and increased to levels higher than those in older children after CPB. Our data show that with standard heparin doses, neonates exhibit less heparin anti-Xa activity during CPB. Higher baseline levels of F1.2 and FPA present in neonates indicate preoperative activation of their coagulation systems as compared with older children. Although F1.2 and FPA levels initially decrease with the commencement of CPB, probably representing hemodilution, the subsequent increase in these markers indicates significantly more thrombin formation and activity during and after CPB. These results raise the concern that 400 U/kg of heparin may not adequately suppress thrombin formation and activity in neonates undergoing CPB.

High antithrombin III levels attenuate hemostatic activation and leukocyte activation during cardiopulmonary bypass

Despite heparinization, cardiac surgery involving cardiopulmonary bypass (CPB) induces a powerful activation of the coagulation and inflammation system. This is caused by the contact of the blood with the large non-endothelial surfaces of CPB, the release and reinfusion of tissue factor-enriched blood from the operation field, the surgical trauma, and the reperfusion of ischemic tissue. As a result, the plasma coagulation factors and platelets are progressively consumed, the complement system and leukocytes are activated, and the proteases and cytokines are released. Clinical consequences of this sequel are hemorrhage, transfusions, and organ damage. Particularly in the myocardium, this systemic activation of the inflammatory system may further trigger regional reperfusion injury and thereby contribute to myocardial stunning, impaired contractility, and low cardiac output. Thrombin plays a pivotal role in the modulation of leukocyte activation: It increases neutrophil recruitment, neutrophil rolling by the expression of endothelial P- and E-selectin, neutrophil adhesion by the expression of intracellular adhesion molecule 1, and the production of endothelial platelet-activating factor. 1 The current investigation assesses the impact of enhanced antithrombin (AT) activity, induced by a high dose of ATIII before the initiation of CPB, on key biochemical markers of hemostatic and leukocyte activation. Methods Patients and anticoagulation protocol. After approval by the local ethics committee and informed consent were obtained, 80 patients undergoing elective coronary artery bypass grafting were enrolled in this prospective, randomized, controlled investigation. No patient received warfarin (Coumadin, Bristol-Myers Squibb, New York, NY) or antiplatelet therapy within 10 days before surgery. Two groups with 40 patients each were formed. In group one (H), in accordance with the departmental standard, anticoagulation was performed according to the results of the Hepcon HMS Plus (Medtronic, Minneapolis, Minn). In group two (HATIII), an additional bolus of 50 IU/kg ATIII (Grifols, Langen, Germany) was given with the initial bolus of heparin before CPB. Blood samples were collected 10 minutes after the administration of the heparin bolus before CPB and after termination of CPB before protamine infusion. Anesthesia was performed using a total intravenous technique with propofol, sufentanil, midazolam, and pancuronium bromide. Normothermic CPB was accomplished with nonheparin-coated systems, membrane oxygenators, and roller pumps. None of the patients received antifibrinolytic agents or the kallikrein inhibitor aprotinin. Laboratory analysis. Heparin anti-Xa activity was determined using the STA-Rotachrom Heparin (Roche, Mannheim, Germany). ATIII was determined using the Coamatic LR Antithrombin Chromogenix assay (Hemochron Diagnostica, Essen, Germany). Prothrombin 1 and 2 fragments (PTF12) were assessed using the Enzygnost F1-2 micro assay (Dade Behring, Marburg, Germany). Soluble fibrin was measured using the COATEST Soluble Fibrin assay (Chromogenix, Milano, Italy). Interleukin (IL)-6 was measured using the IL-6 enzyme-linked immunosorbent assay (DPC Biermann, Nauheim, Germany). Polymorphonuclear leukocyte (PMNL) elastase was determined with the use of a luminescence immunoassay (Auto-CliniLumat LB 952, Bertold, Wildbad, Germany).

Rinsing Dialyzers without Heparin in Hemodialysis (HD) with Low-Molecular Weight Heparin (LMWH)

Saline plus unfractionated heparin (UFH) is routinely used for priming dializers before HD start. The aim of this study is to assess if heparin is necessary in pre-rinsing procedure of extracorporeal systems, anticoagulated with LMWH. We carried-out a prospective study in 14 stable HD patients, aged 58.5 ± 3.6 years old. All patients were treated by standard bicarbonate HD, 3 sessions × week, HD time 220 ± 12 min, Qb 276 ± 21 ml/min, low-flux dialyzers (polisulfone 5, cellulose diacetate 9). Clotting was prevented with a single bemiparin (LMWH) bolus 2900 ± 140 IU. Three rinsing protocols were applied in three consecutive weeks: 1st week- Saline 1000 ml + LMWH 2500 IU. 2d week- Saline 1000 ml + UFH 5000 IU. 3th week- Saline 1000 ml. Anti-Xa activity (IU/ml) was measured at each HD (at 10 min/end). Clotting events and post-HD AVF hemostasis were recorded in a semiquantitative scale. No significant statistical differences (Anova) were found in anti-Xa heparin activity (IU/ml) among the three rinsing protocols tested: 10 min (NS) End (NS) 1st week 0.81 ± 0.16 0.55 ± 0.43 2nd week 0.76 ± 0.21 0.58 ± 0.16 3th week 0.86 ± 0.14 0.64 ± 0.16 No differences were observed in drip chamber or dialyzer appearance after HD. Massive clotting was not recorded. No important bleeding was observed at stick needle sites. In conclusion, HD performed with LMWH no needs heparin in rinsing solution. The procedure is effective and safe.

Evidence of a selective free radical degradation of heparin, mediated by cupric ion

A free radical reaction generated by a mixture of Cu 2+, hydrogen peroxide, and ascorbate causes an abrupt reduction in the anti Xa activity of heparin by about one-half, and in molecular weight by about one-third. The product, which has the characteristics of a “low molecular weight” heparin, differs little in constitution from the intact heparin, on the basis of NMR evidence that includes data for fractions of the polymers. The free radical attack appears to occur adjacent to, rather than directly upon, some residues of α- l-iduronic acid 2-sulfate. Substitution of the Cu 2+ with Fe 2+, results in a less selective alteration of the heparin. Dermatan sulfate undergoes more extensive degradation than heparin with the Cu-reagent, although its anti Xa a potency is less drastically reduced. Overall, the results are more consistent with a high degree of regioselectivity in the interaction between heparin and Cu 2+ ion, than with a delocalized counter-ion interaction.

Efficacy and toxicity of differently charged polycationic protamine-like peptides for heparin anticoagulation reversal.

The role of total cationic charge of synthetic protamine-like peptides in heparin anticoagulation reversal and accompanying adverse hemodynamic effects was studied. Five protamine variants having specific total charges of [+8], [+16], [+18], [+20], and [+21] were synthesized by fluorenylmethoxycarbonyl procedures. Each of these lysine-containing peptides plus arginine-containing control salmine native protamine (n-protamine, [+21] charge) was studied in five dogs who received heparin 150 IU/kg intravenously followed by 1.5 mg/kg (intravenously during a 10-second period) of the synthesized peptide or control n-protamine. Anticoagulation reversal as assessed by a number of coagulation tests was more effective with peptides of greater cationic charge. In this regard, activated clotting time reversal 3 minutes after peptide administration was 7%, [+8]; 54%, [+16]; 81%, [+18]; 92%, [+20]; 81%, [+21]; and greater than 100% [n-protamine]. Reversal of heparin anticoagulation at 3 and 30 minutes, respectively, correlated significantly (*p < or = 0.05, p < or = 0.01 [see corresponding symbols within abstract]) with total cationic charge as assessed by activated clotting time (r = 0.97, 0.99 ), prothrombin time (r = 0.98, 0.87*), activated partial thromboplastin time (r = 0.99, 0.78), thrombin clotting time (r = 0.84,* 0.85*), heparin anti-Xa activity (r = 0.87,* 0.85*), and heparin anti-IIa activity (r = 0.79 at 3 minutes, p = 0.06). Maximum declines in systemic mean arterial pressure (MAP) were greater with more positively charged peptides: -1 mm Hg, [+8]; -3 mm Hg, [+16]; -31 mm Hg; [+18]; -31 mm Hg, [+20]; -35 mm Hg, [+21]; and -34 mm Hg [n-protamine]. Maximum decreases in MAP, cardiac output, and systemic oxygen consumption were highly correlated (p < or = 0.05) with total cationic charge: MAP, r = 0.87; cardiac output, r = 0.87; and systemic oxygen consumption, r = 0.86. A total toxicity score, reflecting adverse hemodynamic effects, was greater with increasing charge: -1.9 +/- 1.1, [+8]; -2.7 +/- 0.8, [+16]; -6.6 +/- 3.3, [+18]; -6.1 +/- 3.5, [+20]; -6.9 +/- 3.8, [+21]; and -7.0 +/- 5.2 [n-protamine]. The correlation of mean peptide total toxicity score to total cationic charge was significant (r = 0.89, p < 0.05). These data suggest for the first time that effective alternatives to salmine protamine for reversal of heparin anticoagulation can be synthesized. Furthermore, total cationic charge appears to be an important determinant for both anticoagulation reversal and toxicity of protamine-like peptides.