Factor Xa Inhibition Assay Research Articles

Structural alteration in hypochlorous acid modified antithrombin indicates generation of neo-epitopes

Increased tendency of cancer patients to develop venous thromboembolism (VTE) is associated with high rates of mortality. Elevation of procoagulant proteins and down regulation of naturally occurring coagulation inhibitors appears to form the basis of high risk of VTE in malignancy. A reduced level of anticoagulant protein like antithrombin (AT) will influence both coagulation and angiogenesis, as its cleaved and latent conformations show potent antiangiogenic activity. We show a concentration dependent perturbation in the secondary and tertiary structures of AT conformers exposed to hypochlorous acid (HOCl). Modulated under a very narrow concentration range of HOCl, native AT undergoes oligomerization, aggregation and fragmentation based on spectroscopic, SDS and native-PAGE studies. Factor Xa inhibition assay demonstrated a progressive decrease in inhibition activity of AT on modification by HOCl. Bis-ANS result showed that hydrophobic patches were more exposed in the case of HOCl-modified AT when assessed fluorometrically. Dosage of HOCl-modified AT in experimental animals induced high titer antibodies showing more specificity towards modified forms in comparison to unmodified forms. Auto-antibodies isolated from cancer patients also showed enhanced binding with HOCl-modified AT in comparison to native counterpart. Compared to normal AT, structurally and functionally altered conformation of HOCl-modified AT showed increased immunogenic sensitivity. HOCl modified AT can contribute to prothrombotic and angiogenic environment during cancer progression/development.

A nonelutable low-molecular weight heparin stent coating for improved thromboresistance

Low-molecular weight heparin (LMWH) has been widely used as a systemic anticoagulant during percutaneous coronary intervention. In this study, LMWH was covalently immobilized to the surface of a cobalt chromium reservoir-based sirolimus-eluting stent to create a nonelutable nanoscale coating for enhanced thromboresistance. Toludine-blue stained stents revealed uniform heparin coverage on all surfaces of the stent. Scanning electron microscopy of stent strut cross-sections showed identical coating thickness on all sides; while the thickness was determined to be 320 nm by a focus-ion beam system. Secondary ion mass spectrometry showed constant concentrations of O, N, and S atoms throughout the depth of the surface, confirming the uniformity of the heparin coating. The nonelutable nature of the coating was confirmed in a modified Factor Xa inhibition assay which showed the stent had an equivalent of 3-5 heparin units/cm(2), while no elutable heparin was detected in wash solutions. The antithrombin binding capacity of the immobilized heparin was determined to be 60-80 pmol/cm(2) in an antithrombin uptake assay. The enhanced thromboresistance of the heparin coating was demonstrated in an in-vitro bovine blood flow loop which showed minimal visual thrombus accumulation and 95% reduction in platelet deposition compared to uncoated control stents. Drug-eluting stents with such nonelutable LMWH coating would represent a significant advance in the treatment of patients with complex lesions who are at increased risk of developing stent thrombosis.

Interlaboratory Precision in the Monitoring of Unfractionated Heparin Using the Anti-Factor Xa-Correlated Activated Partial Thromboplastin Time

Although the activated partial thromboplastin time (aPTT) remains the most widely used method for monitoring unfractionated heparin (UFH) therapy, it is affected by a number of preanalytic, analytic, and biological variables, which undermine both its accuracy and precision. In an effort to improve the accuracy and precision of laboratory monitoring of UFH, the College of American Pathologists (CAP) and the American College of Chest Physicians (ACCP) have issued guidelines recommending that the therapeutic range of the aPTT be defined in each laboratory through correlation with a direct measurement of heparin activity such as the factor Xa inhibition assay (anti-FXa). Whether and to what extent this approach enhances the precision of UFH monitoring has not been reported. We conducted a cross-validation study among 4 CAP-accredited coagulation laboratories to assess the interlaboratory precision of the anti-FXa-correlation method. An aPTT and anti-FXa were performed in each laboratory on plasma samples from 44 inpatients receiving UFH. Interlaboratory precision of the anti-FXa-correlation method was compared to that of the traditional 1.5–2.5 times the upper limit of normal (ULN) method for defining the therapeutic aPTT range. Modest to poor intralaboratory correlation between the aPTT and anti-FXa was observed in each of the 4 laboratories. The coefficients of determination (R2) ranged from 0.1962 to 0.6964. In accordance with CAP guidelines, the anti-FXa-derived therapeutic aPTT range was defined by linear regression corresponding to a range of anti-FXa activity of 0.3 – 0.7 units/ml. In each laboratory, the range defined by this method was broader than that defined using the ULN method. In 3 of the laboratories, the therapeutic range defined by the anti-FXa-correlation method extended more than 20 seconds beyond the upper limit of the therapeutic range defined by the ULN approach. Based on the laboratory-specific therapeutic ranges defined by both methods, samples were segregated into therapeutic category [i.e. below therapeutic (BT), therapeutic (T), or above therapeutic (AT)]. Using the ULN method, there was agreement among all 4 laboratories regarding the therapeutic category in 22 (50%) samples, whereas consensus was achieved in only 7 (16%) samples with the anti-FXa-correlation method. Furthermore, 3 (7%) samples were simultaneously determined to be BT and AT in different laboratories by the anti-FXa-correlation method, suggesting that the dose of UFH might be increased in one center and decreased in another for the same patient encounter. This striking discrepancy was not observed with the ULN method. In conclusion, the anti-FXa-correlation method for defining the therapeutic range of the aPTT does not enhance the interlaboratory precision of UFH laboratory monitoring and may be inferior to the ULN method in this regard. Clinical studies are needed to assess the impact of these findings on patient safety.

Interlaboratory agreement in the monitoring of unfractionated heparin using the anti-factor Xa-correlated activated partial thromboplastin time.

In an effort to improve interlaboratory agreement in the monitoring of unfractionated heparin (UFH), the College of American Pathologists (CAP) recommends that the therapeutic range of the activated partial thromboplastin time (APTT) be defined in each laboratory through correlation with a direct measure of heparin activity such as the factor Xa inhibition assay. Whether and to what extent this approach enhances the interlaboratory agreement of UFH monitoring has not been reported. We conducted a cross-validation study among four CAP-accredited coagulation laboratories to compare the interlaboratory agreement of the anti-FXa-correlated APTT with that of the traditional 1.5-2.5 times the midpoint of normal (1.5-2.5:control) method for defining the therapeutic APTT range. APTT and FXa inhibition assays were performed in each laboratory on plasma samples from 44 inpatients receiving UFH. Using the anti-FXa-correlation method, there was agreement among all four laboratories as to whether a sample was subtherapeutic, therapeutic or supratherapeutic in seven (16%) patient samples. In contrast, consensus was achieved in 23 (52%) samples when the 1.5-2.5:control method was employed. The anti-FXa-correlation method does not appear to enhance interlaboratory agreement in UFH monitoring as compared with the traditional 1.5-2.5:control method. Adoption of the anti-FXa-correlation method produces considerable disparity in UFH dosing decisions among different centers, although the clinical impact of this disparity is not known.

Insight into the mechanism of asparaginase-induced depletion of antithrombin III in treatment of childhood acute lymphoblastic leukemia

Asparaginase (ASNase) is a widely used and successful agent against childhood acute lymphoblastic leukemia (ALL). Asparaginase cleaves asparagine (Asn) to give aspartic acid and ammonia, thereby depleting free Asn in the blood. However, treatment with ASNase has been implicated in significant reduction of plasma levels of the coagulation serine protease inhibitor (serpin) antithrombin III (AT3), predisposing patients to thromboembolic complications. Our investigation was designed to delineate the biochemical mechanism of AT3 depletion that can occur in the plasma of ALL patients undergoing ASNase therapy. SDS-PAGE showed no cleavage of purified AT3 following treatment with ASNase. Furthermore, purified AT3 treated with ASNase demonstrated no decrease in inhibitory activity. Human plasma and whole blood treated with approximate therapeutic concentrations of ASNase showed no loss of AT3 activity as detected by a plasma-based factor Xa inhibition assay. Treatment of a confluent monolayer of HepG2 (hepatocarcinoma) cells with ASNase showed no gross loss in AT3 message levels detected by rtPCR. However, a decrease of cell viability was observed in cultures treated with ASNase. Interestingly, medium from HepG2 cells treated with ASNase showed a marked decrease in secretion of AT3 and another serpin, heparin cofactor II. Collectively, these data show that ASNase has no direct effect on AT3 in blood or plasma, but that ASNase may affect plasma levels of AT3 by interfering with translation and/or secretion of the protein in liver cells.

Heparin detection by the activated coagulation time: A comparison of the sensitivity of coagulation tests and heparin assays

Objective : Laboratory and point-of-cared coagulation tests are frequently obtained to determine the presence of heparin after surgical procedures. The objective of this study was (1) to compare the sensitivity of the activated coagulation time (ACT), activated partial thromboplastin time (aPTT), protamine titration (Hepcon; HMS Medtronic, Hemotec, Englewood, CO), and thromboelastography (TEG) with heparin anticoagulation and (2) to determine how frequently residual heparin is present in the 24-hour period after heparin neutralization in cardiopulmonary bypass (CPB) patients. Design : A prospective study. Setting : A tertiary care university teaching center that performs more than 15,000 surgical procedures per year. Participants : Vascular surgical ( n = 17) and CPB ( n = 29). Interventions : In vascular surgical patients, coagulation tests (ACT, protamine titration [Hepcon], and TEG) were obtained before and 90 minutes after heparin (50 to 60 U/kg IV) and compared with heparin concentration determined by factor Xa inhibition assay. In cardiac surgical patients, ACT and heparin concentrations were measured after anesthesia induction, during CPB, after protamine neutralization, and 3 as well as 6 hours after CPB. In addition to heparin concentrations and ACT measures, platelet counts, fibrinogen levels, and bleeding times were determined before and 3 and 24 hours after CPB. Measurements and Main Results : Ninety minutes after heparin, significant heparin concentrations were present in all vascular surgical patients, but ACT was elevated only 4 of 17 patients. Protamine titration (Hepcon) correlated with the factor Xa inhibitory assay for heparin ( r 2 = 0.76). All 17 patients had an abnormal TEG (mean “R” time = 81 ± 39 minutes) and a marked elevation of aPTT (135 ± 35 sec [normal 22 to 33 seconds]) 90 minutes after heparin. In CPB patients, ACT did not correlate with heparin assays. After protamine neutralization of heparin in CPB patients, aCT returned to baseline despite the presence of heparin in 3 of 29 patients (0.22, 0.18, and 0.33 U/mL). Conclusions : ACT was les sensitive to residual heparin anticoagulation than aPTT, TEG, and whole blood heparin assay. The whole blood heparin assay (Hepcon) provided sensitive and specific data about the presence of residual heparin. Despite the limitation of ACT in detecting heparin, the investigators found that residual heparin was not common in the period after uncomplicated CPB.

Prevention of fetal death in the antiphospholipid antibody syndrome.

The first treatment of pregnant women with antiphospholipid antibody syndrome (APLS) employed high doses of corticosteroids, plus low dose aspirin, with the goal of suppressing production of the autoantibody. Corticosteroids (usually prednisone), even when much lower doses are used, and even when tapered after midpregnancy, have been associated with significant maternal and obstetric risks and side effects: the most important are osteomalacia and preterm delivery (often precipitated by premature rupture of the membranes). Since the publication of a randomized trial demonstrating equivalent live birth rates of about 75% whether heparin or prednisone was used for treatment (plus low dose aspirin), the use of adjusted doses of heparin, together with low dose aspirin, has replaced prednisone for treatment of pregnant women; although prednisone may still be needed to treat manifestations of associated autoimmune disorders. A recent randomized trial has shown that the addition of heparin to aspirin is probably superior to treatment with aspirin alone. To achieve prophylactic levels of plasma heparin equivalent to those measured in patients who are not pregnant and are treated with the usual dose of standard heparin of 5000 IU every 12 h, the heparin dose required for treatment of pregnant women is usually higher. For that reason, heparin doses should be adjusted using the nadir APTT, or better plasma heparin measured by a factor Xa inhibition assay at the 2 h post-injection peak. Although low molecular weight heparin has been shown to be useful in prevention of fetal resorption in a mouse model, and appears to be equally safe for treatment of pregnant women, we still have no published data to show therapeutic equivalency, with respect to treatment of APLS-complicated pregnancy, to standard heparin preparations, and none that demonstrate any lower risk for the complication of most concern when heparin is given to pregnant women-osteopenia. Similarly, intravenous infusion of gamma globulins (IVG) appears on the basis of case reports to be effective additional treatment in cases where standard therapy has failed. Gamma globulin preparations contain anti-idiotypic antibodies that have been shown to bind to patient antiphospholipid antibodies. The place for the addition of IVG to standard therapy has not been defined, but clinically significant and corticosteroid-resistant thrombocytopenia complicating antiphospholipid antibody syndrome might be one indication for primary treatment with IVG +/- low dose aspirin. Overall, live birth rates in most treatment studies are in the range of 70-80%. The reported birth rate information, however, cannot be compared between studies. None of the studies reported have used tools such as logistic regression analysis to allow for such significant predictors of live birth as the number of prior miscarriages, maternal age, medical history, or a history of fetal death (loss of a viable and chromosomally normal fetus after the 10th gestational week).

Isolation and characterization of recombinant annexin V expressed in Saccharomyces cerevisiae

The cloning, purification and characterization of full-length annexin V, expressed intracellularly in Saccharomyces cerevisiae is detailed. Following homogenization in a glass bead mill, clarification by ultracentrifugation and fractional ammonium sulfate precipitation, the 319 amino acid protein was purified by column chromotography on phenyl-Sepharose and heparin-Sepharose. Annexin V elutes on reverse phase C 4 silica as a single peak with greater than 97% homogeneity and is further characterized by a molecular mass of 34 kDa from electrophoresis under reducing conditions on SDS gels. Dynamic light scattering experiments reveal annexin V exists as a monomer in solution. Amino terminal Edman degradation afforded no sequence, therefore the carbamidomethylated protein was chemically cleaved with cyanogen bromide. Separation of the resulting peptide fragments on reverse phase HPLC followed by N-terminal sequencing and electrospray mass spectrometry supported the correct sequence as well as the existence of an acetyl blocking group on the N-terminus. The protein exhibits an isoelectric point of 4.73 by column chromatofocusing. Secondary structure predictions from CD spectroscopy indicate that the molecule is correctly folded. In anticoagulant assays, the purified protein exhibits dose-response effects in activated partial thromboplastin time (APTT) prolongation and doubles the clotting time of control human plasma at 70 μg ml −1. More specifically, in a factor Xa inhibition assay in which the activation of factor X via the tissue factor-factor VIIa complex is monitored by the cleavage of a factor Xa chromogenic substrate, recombinant annexin V exhibits a 50% inhibitory concentration (IC 50) in the low nanomolar range.

Increased concentrations of heparin cofactor II in diabetic patients, and possible effects on thrombin inhibition assay of antithrombin III.

We compared concentrations of antithrombin III (AT-III) in plasma, as determined by an immunological method and by a functional thrombin inhibition method, in the presence of heparin in 160 blood samples from Type I diabetics. Although the correlation was highly significant (P less than 0.001) between the results obtained by the two methods, our data demonstrated that results by the thrombin inhibition assay, 121 (SD 15)%, expressed as percentages of the results for a normal plasma pool, were significantly (P less than 0.001) higher than by the immunoreactive method, 104 (SD 15)%, indicating an overestimation of functionally active AT-III. Concentrations of functionally active AT-III determined by a factor Xa inhibition assay, 105 (SD 13)%, were in the same range as immunoreactive AT-III. Addition of IgG antiserum to normal pooled plasma quenched only about 90% of the AT-III activity determined by the thrombin inhibition assay, but all of the AT-III activity determined by a factor Xa inhibition assay. These results demonstrate that the factor Xa inhibition assay is more specific for the determination of AT-III than the thrombin inhibition assay. We suggest that the high concentrations of heparin cofactor II, 117 (SD 17)%, might have caused an overestimation of AT III in this group of patients with diabetes Type I, and should not be overlooked in other clinical situations.

Circadian changes in anticoagulant effect of heparin infused at a constant rate.

Six patients with venous thromboembolism were treated with heparin, administered intravenously by a constant infusion pump. The initial daily dose of heparin was adjusted to keep the activated partial thromboplastin time, sampled at 0800, between 1.5 and 2.5 times the control level. Once that level was obtained, this dose was kept constant. Anticoagulation was thereafter measured, every four hours for 48 hours, by activated partial thromboplastin time, thrombin time, and coagulation factor Xa inhibition assay. The results of all three coagulation tests showed a circadian variation in the six patients. Maximum values were achieved at night and minimum values in the morning. These circadian variations were reproduced for two consecutive days. Differences between night and morning values reached almost 50% for activated partial thromboplastin time, 60% for thrombin time, and 40% for factor Xa inhibition assay. This circadian variation resulted from two rhythms, a circadian rhythm lasting 24 hours and an ultradian rhythm lasting 12 hours, which were detected by cosinor analysis for each coagulation test (p less than 0.01). A circadian rhythm was detected individually in most of the patients for each coagulation test (p less than 0.05). All patients had a nocturnal peak in activated partial thromboplastin time on both days. In four patients this peak exceeded the upper desired limit of activated partial thromboplastin time. These rhythms should be taken into account when evaluating the dosage of heparin to be administered.

Chromatography of heparin on sepharose-lysine: molecular size fractionation and its relevance to thrombin and antithrombin III binding.

Batches of Sepharose–lysine, which varied in lysine content from 35 to 430 μmol/g of dry gel, were prepared by varying the quantity of CNBr in the activation reaction. The batches were tested for heparin binding by using a controlled chromatographic procedure. Sepharose–lysine, containing < 150 μmol of lysine/g, did not significantly bind heparin whereas conjugates with > 400 μmol/g retained the entire heparin load. For intermediate batches of Sepharose–lysine (150–400 μmol/g) the quantity of heparin bound largely paralleled the lysine content. Thus, Sepharose–lysine of an intermediate lysine content separated heparin into an unretained fraction and a bound fraction which was recovered from the column by eluting with 1 M NaCl. On testing for anticoagulant activity by factor Xa inhibition assay, no significant difference in specific anticoagulant activity was observed between these heparin fractions and the heparin load. However, from gel filtration studies, a substantial difference in molecular size was noted. An unretained heparin fraction from Sepharose–lysine was of a lower average molecular weight than the parent heparin. In contrast, a retained heparin peak was of a higher average molecular weight compared with the parent heparin. These observations were confirmed by studying the chromatographic properties of low (10 000) and high (23 000) molecular weight heparin samples on various Sepharose–lysine batches. A model is proposed to explain this discriminating property of Sepharose–lysine. For a conjugate containing 400 μmol/g, the mean lysine spacing is calculated at 47 Å (1 Å = 0.1 nm), which is approximately equivalent to five to six disaccharide units in heparin.The property of Sepharose–lysine to bind heparin was compared with the affinities of the mucopolysaccharide for both thrombin and antithrombin III. Evidence has been proposed for the involvement of lysine residues of antithrombin III in this process. Our investigations suggest that lysine, in addition to arginine, groups of thrombin are also involved in heparin binding. By specifically modifying two to four lysine residues using nitrous acid, the heparin-binding capacity of the enzyme and its plasma clotting activity were largely destroyed, although the esterase activity was retained.