Clinical monitoring of hirudin and direct thrombin inhibitors.

In addition to heparin, the standard medication for prophylaxis and therapy of thromboembolism, several other substances have been developed and tested for clinical use with the aim of decreasing or eliminating side effects. Most of all, hirudin, a direct antithrombin (AT), has proved to be effective. To define the therapeutic range and to avoid underdosage or overdosage, clinical monitoring is necessary. For monitoring of hirudin, thrombin time (TT) is not suited because of the missing linearity of the standard curve. Activated partial thromboplastin time (aPTT) can be used only in the lower hirudin level range, where the standard curve is quite linear. However, high and toxic hirudin levels cannot be determined using aPTT. Another drawback is a high variation in single measurements and in the normal value of patients. Methods using chromogenic substrates are suited for determination of hirudin in plasma but cannot be used at bedside. Especially for monitoring of hirudin, the ecarin clotting time (ECT) was developed. The standard curve is linear over the entire concentration range. There are no influences by other coagulation parameters or anticoagulants. For both acute clinical situations and long-term monitoring, this method capable of point-of-care therapy (POCT) will be the method of choice.     

Comparison of three methods for measuring PEG-hirudin in blood.

Three methods for measuring pegylated hirudin (PEG-hirudin), a new antithrombotic agent, in blood were compared using clinical samples. The ecarin clotting time (ECT) was performed in whole blood using a point-of-care device (TAS analyzer). The ECT was also performed in plasma, using a clotting assay in a conventional automated coagulation analyzer. Finally, a chromogenic method was used, based on thrombin inhibition and the substrate S-2238. Both clotting assays showed a linear relationship between the ECT and the PEG-hirudin concentration up to 3.0 microg/ml. The chromogenic substrate method was linear only between 0.1 and 1.0 microg/ml PEG-hirudin. The intra-assay coefficient of variation was 3.0% for the automated ECT method, 6.4% for the point-of-care ECT method and 3.4% for the chromogenic method. The inter-assay coefficient of variation was approximately 10% for both clotting methods and 3.2% for the chromogenic method. There was a high correlation (r = 0.954) in PEG-hirudin concentration between both ECT methods over the entire measuring range. The correlation of the chromogenic method with any ECT was significantly less (r < 0.89), even if only PEG-hirudin concentrations < 1.0 microg/ml were taken into account (r < 0.92). Although we clearly prefer the conventional ECT, any of the other methods may be used for monitoring PEG-hirudin in patients treated with this drug, depending on the specific application and local circumstances.     

Monitoring of anticoagulant effects of direct thrombin inhibitors

Monitoring of direct thrombin inhibitors with the activated partial thromboplastin time (aPTT) is limited by poor linearity and reproducibility. Recently, direct prothrombin activation methods have been developed for coagulation analysis: ecarin clotting time (ECT) and prothrombinase-induced clotting time (PiCT). Laboratory monitoring of the direct thrombin inhibitors lepirudin, argatroban, and melagatran was analyzed and compared with monitoring unfractionated heparin (UFH). Plasma samples of six healthy donors were spiked with lepirudin and argatroban extending to 3000 ng/mL, melagatran extending to 1000 ng/mL, and UFH up to 0.48 IU/mL. Clotting times of aPTT (two reagents), ECT, PiCT, and prothrombin time were determined in a KC 10, a micro instrument. At 3000 ng/mL ECT values were 339.1 +/- 25.0 seconds with lepirudin and 457.5 +/- 29.5 seconds with argatroban. ECT was 586.0 +/- 38.2 seconds with 1000 ng/mL melagatran. The PiCT method provided clotting times of 137.0 +/- 8.4 seconds with UFH, 128.0 +/- 23.4 seconds with lepirudin, and 151 +/- 23.9 seconds with argatroban, and 153.5 +/- 9.9 seconds with melagatran, with the concentrations mentioned. ECT is more sensitive to therapeutic drug concentration ranges than aPTT (prolongations of 3-7 versus 2-3). PiCT yields comparable results with direct thrombin inhibitors and UFH. This method could therefore be suitable for monitoring both drug groups.     

A functional clotting assay to monitor the hirudin dosage.

Hirudin, a direct thrombin inhibitor, has potential advantages over indirect thrombin inhibitors and is increasingly used in clinical settings. There are, however, large variations in individual responses to this drug and no recognized clinical laboratory tests used to monitor its anticoagulant effects. We evaluated the use of the thromboelastograph, a common clinical coagulation instrument, to monitor the effects of hirudin in vitro. We developed a novel, whole blood clotting assay that utilizes the tissue factor stimulating properties of mercuric ion to measure the anticoagulant potential of therapeutic doses of hirudin. At doses equivalent to those found in the therapeutic range, the thromboelastograph was capable of showing significant changes when compared with control and different concentrations of hirudin (P < 0.05). A linear relationship was observed between increasing concentrations of recombinant hirudin and clotting times. In conclusion, the use of this test system warrants further investigation for monitoring hirudin.     

A step change in oral anticoagulation: lack of coagulation monitoring with ximelagatran

The clinical development of ximelagatran for the treatment and prevention of various arterial and venous thromboembolic disorders has used fixed-dose regimens without coagulation monitoring in all indications. Although monitoring is not required, effects on the various coagulation assays that are available are seen with its active form melagatran, and there are situations where an assessment of anticoagulant effect may help to inform clinical decisions. However, the sensitivity of different coagulation assays varies considerably. The thrombin clotting time (TT) and ecarin clotting time (ECT) are highly sensitive to plasma melagatran concentrations (IC 50 approximately 0.01 micromol/L and approximately 0.15 micromol/L, respectively), with an approximate linear relationship between plasma melagatran concentration and prolongation of clotting time. In comparison, the activated partial thromboplastin time (APTT) (IC 50 approximately 0.3 to 0.8 micromol/L) and prothrombin time (PT) (IC 50 approximately 0.9 to 2.9 micromol/L) are relatively insensitive, and the concentration-response relationship shows a flattening with increasing plasma melagatran concentration. Commercially available APTT and PT reagents varied considerably in their sensitivity to melagatran. Comparing the various coagulation assays, the APTT, ECT, and TT are suitable choices when an indicator of the anticoagulant effect of ximelagatran is required, although the absence of international standards requires calibration of each test in individual laboratories and the ECT is not widely available.     

Determination of antithrombin-dependent factor Xa inhibitors by prothrombin-induced clotting time

Prothrombinase-induced clotting time (PiCT) determines the anticoagulant effects of heparins, low molecular weight heparins (LMWHs), and direct thrombin inhibitors. At present, this is the only method that measures the effects of all of these inhibitors, in contrast to the prothrombin time, activated partial thromboplastin time (aPTT), Heptest, ecarin clotting time, and the chromogenic assays. The antithrombin-dependent direct factor (F) Xa inhibitors fondaparinux and idraparinux were compared with the LMWH dalteparin on PiCT, aPTT, Heptest, and chromogenic anti-FXa assays in pooled human normal plasma samples. Fondaparinux and idraparinux prolonged the coagulation times in the PiCT, Heptest, and chromogenic FXa assays in a dose-dependent manner, in contrast to the aPTT. We conclude that PiCT is a suitable assay to determine the anticoagulant effects of these two new FXa inhibitors in patients receiving treatment with these compounds.     

Monitoring the new antithrombotic drugs

Today there is a diverse group of anticoagulant and antithrombotic drugs available that includes warfarin derivatives, heparin, low-molecular-weight heparins, thrombin inhibitors, factor Xa inhibitors, and various antiplatelet agents. Many of these new drugs do not alter measurable blood coagulation parameters, yet they are effective antithrombotic agents through their actions on vascular endothelial cells and proteins. Thus, these new agents do not affect the traditional clot-based prothrombin time/International Normalized Ratio (PT/INR) and activated partial thromboplastin time (aPTT) tests, and monitoring and standardization require the development of new methods. In addition to clot-based assays, chromogenic assays, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), flow cytometry, and other techniques have been used to monitor these new drugs. On the other hand, some of the new antithrombotic drugs do affect the PT, aPTT, and activated clotting time (ACT); however, they behave differently from the warfarin derivatives and heparin. The traditionally used relationship of target time to clot values and INR to clinical effect cannot necessarily be transferred to the new drugs. Unfortunately, monitoring is not as simple as it was for warfarin and heparin. Although the new antithrombotic drugs have been approved for clinical use, assay systems for monitoring most of them are still in development or have not been clinically validated. This applies to each of the clinical settings targeted for prophylaxis, treatment, or interventional procedures (i.e., high- and low-dosing regimens typically require different monitoring methods). In addition to basic monitoring, other issues such as sensitivity of the drug to different laboratory monitoring reagents and instrumentation, drug combination monitoring, and patient-related factors that contribute to the variability of the results still need to be addressed.     

Determination of the anticoagulant effects of new oral anticoagulants: an unmet need

Thromboembolic diseases require anticoagulation for their prevention and treatment. New oral anticoagulants, specifically direct factor Xa and thrombin inhibitors, were developed to overcome the limitations of conventional anticoagulants. Their benefit has been demonstrated using fixed doses without laboratory-guided dose adjustment for patients following elective knee and hip replacement, treatment of venous thromboembolism and prevention of embolic events in atrial fibrillation. These anticoagulants are excreted by glomerular filtration at a rate of between 25 and 80%. Thus, lower doses are required for patients with impaired renal function. Therefore, determination of the anticoagulant effects may be needed in other specific patient populations. Prothrombin time, activated partial thromboplastin time, prothrombin-induced clotting time, ecarin clotting time, hemoclot assay, other specific coagulation assays and chromogenic substrate are available to determine the effect of the anticoagulants. Standardization of methods, development of point-of-care tests and identification of patient groups is ongoing.     

Measurement of antithrombin activity by thrombin-based and by factor Xa-based chromogenic substrate assays.

Functionally active antithrombin can be quantified by chromogenic substrate assays utilizing the heparin cofactor activity of antithrombin and the inhibition rates of thrombin or of activated factor X (FXa). Thrombin-based assays but not FXa-based assays may overestimate the antithrombin activity due to their sensitivity toward heparin cofactor II. We focused on the question whether an overestimation of antithrombin activity by thrombin-based assays involves the risk of misdiagnosing antithrombin-deficient individuals as being non-deficient. We determined antithrombin using two thrombin-based assays and one FXa-based assay in 27 plasma samples from patients with acquired antithrombin deficiency spiked with lepirudin, in antithrombin-deficient plasma and in mixtures of antithrombin-deficient plasma and normal plasma. We also measured antithrombin in healthy subjects, in patients with inherited and acquired antithrombin deficiency and in patients under high-dose heparin treatment. At therapeutic final concentrations of lepirudin, antithrombin activities were considerably overestimated by the thrombin-based assays but not by the FXa-based assay. The residual antithrombin activities in antithrombin-deficient plasma determined by the thrombin-based assays were markedly higher than the corresponding values obtained with the FXa-based assay. The thrombin-based assays also overestimated antithrombin activity in patients under high-dose heparin. However, the degree of overestimation in the range between 50 and 100 IU/dl was too low to misidentify individuals with inherited or acquired antithrombin deficiency as normal. We conclude that functionally active antithrombin can be reliably determined using FXa-based chromogenic substrate assays in all settings examined. Thrombin-based assays must not be used in patients under treatment with hirudin or other direct thrombin inhibitors.     

Hirudin in renal insufficiency

Recombinant hirudins (r-hirudins) are potent direct thrombin inhibitors increasingly used for alternative anticoagulation, especially in heparin-induced thrombocytopenia. R-hirudins are almost exclusively eliminated by the kidneys, and a close correlation between r-hirudin clearance and endogenous creatinine clearance has been observed. Accordingly, the pharmacokinetics of r-hirudin are altered in patients with renal insufficiency. A decline of renal r-hirudin clearance is associated with an increase of r-hirudin half-life and the area under the curve (AUC). Therefore, renal impairment necessitates reduction of r-hirudin dose to avoid overdose or inadequate accumulation of the thrombin inhibitor. To this end, close monitoring of r-hirudin anticoagulation is required, which at best is performed by measuring r-hirudin blood levels by ecarin clotting times (ECT) or chromogenic assays, in addition to activated partial thromboplastin time (aPTT). Recent studies showed that r-hirudin anticoagulation is feasible in acute or chronic renal failure treated with continuous or intermittent renal replacement therapy, if appropriate r-hirudin dosing and adequate monitoring are warranted. High-volume hemofiltration with r-hirudin-permeable hemodialyzers constitutes a valuable means to markedly reduce r-hirudin blood concentration and total r-hirudin body content in case of r-hirudin overdose or r-hirudin-associated bleeding. In the future, the hepatically eliminated direct thrombin inhibitor argatroban may facilitate alternative anticoagulation in patients with renal insufficiency.     

Use of a centrifugal analyzer for a chromogenic prothrombin time, a chromogenic activated partial thromboplastin time and a kinetic fibrinogen assay in a routine hospital laboratory

A Cobas Bio centrifugal analyzer was used in a clinical laboratory for the performance of chromogenic clotting assays. Three commercially available photometric clotting tests--prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen--were compared with the traditional clotting assays during 3 months. No great discrepancies were found between the traditional assays and the new photometric assays. The chromogenic PT could replace the traditional thrombotest, PT and Normotest, because it was sensitive and accurate over a broad range of clotting factor activity. Furthermore the chromogenic PT could be used to discriminate between a decreased clotting activity due to vitamin K deficiency or to a decreased protein synthesis by the liver. A decreased protein synthesis was confirmed by measuring a decrease in the serum cholinesterase activity. The chromogenic aPTT could be used for the assay of heparin concentrations in the therapeutic range and turned out to be more sensitive for deficiencies of factor VIII and factor IX than a traditional clotting aPTT. We conclude that the accuracy and practicability of clotting assays are improved by the new assays without diminishing the clinical value of the results.     

Comparative studies on various assays for the laboratory evaluation of r-hirudin.

Several laboratory methods are available to measure r-hirudin, including clot-based, amidolytic, immunologic, and physicochemical techniques. The global tests, such as the PT, APTT, and Heptest, did not show an adequate response to r-hirudin in the range of 0.5 to 10.0 micrograms/ml, where full anticoagulation is achieved, as determined by animal models of thrombosis. The 10 U/ml thrombin time assay was very sensitive to r-hirudin, whereas the 10 U/ml calcium thrombin time gave a dose-dependent response from 0.15 to 10.0 micrograms/ml. Whole blood clotting assays (ACT, TEG) effectively measured r-hirudin levels up to 25 micrograms/ml. The amidolytic anti-Factor IIa assay, specific for evaluating direct thrombin inhibition, was very effective, particularly when modified to decrease the sample: thrombin ratio for higher r-hirudin concentrations. This assay may be useful in quality control, since it is biochemically defined and reagents are easily standardized. Thrombin generation assays based on synthetic substrates showed limited effect of r-hirudin; however, assays based on TAT complex and prothrombin fragment F1+2 generation showed a dose-dependent response. Immunologic methods (ELISA) are under development. Since these assays measure both complexed and noncomplexed hirudin, and since they are only sensitive to submicrogram levels, they may only be useful for the direct quantitation of absolute levels of r-hirudin but not for monitoring clinical anticoagulation. Thus, thrombin-based clotting, amidolytic, and immunologic assays can be used to evaluate and measure r-hirudin. However, optimization of each assay to respond to high and low concentrations of r-hirudin and their application to clinical monitoring, batch control, and standardization needs to be determined.     

Laboratory measurement of the direct oral anticoagulants

Direct oral anticoagulants (DOACs), including the direct thrombin inhibitor, dabigatran, and the direct factor Xa (FXa) inhibitors, rivaroxaban, apixaban and edoxaban, are approved for thromboembolism prevention and treatment. These drugs do not require routine coagulation monitoring but, in some circumstances, measurement of drug level or anticoagulant effect may be necessary. Although traditional coagulation tests lack analytical sensitivity and specificity, they are widely available and inexpensive, and can provide useful information regarding the residual anticoagulant effect of DOACs. Hemoclot^® and ecarin‐based assays can be used to quantify dabigatran level and calibrated chromogenic anti‐FXa assays are suitable for measuring rivaroxaban, apixaban and edoxaban levels, but these tests are not yet widely available.     

Hirudin in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT), a serious side effect of heparin treatment, requires alternative anticoagulation in most affected patients. The recombinant hirudin (r-hirudin) lepirudin has been approved for this purpose after two prospective trials in laboratory-confirmed HIT patients. Other drugs available for this purpose are danaparoid sodium (a heparinoid) and argatroban, a synthetic direct thrombin inhibitor. In this article, recommendations for optimal use of r-hirudin in HIT are given, covering therapy in uncomplicated patients as well as in special situations such as heparin reexposure of HIT patients. Because lepirudin's half-life depends on renal function, it may vary between 1 and 200 hours, which requires individual dose adjustments. Lepirudin compares favorably with danaparoid, based on retrospective data. No direct comparisons of lepirudin with argatroban are available, but argatroban might offer advantages in patients with renal failure, because it is mainly eliminated hepatically. Major hemorrhage, the main risk of lepirudin treatment, occurring in about 15% of patients, makes close monitoring important. New monitoring tools, such as the ecarin clotting time (ECT), might further reduce bleeding risks. Antihirudin antibodies, which can alter the pharmacokinetics as well as the pharmacodynamics of hirudin, can also be countered by close monitoring and appropriate dose adjustments. Whereas hirudins have not yet managed to gain importance in non-HIT indications such as unstable coronary syndromes, they have a major role to play in the treatment of HIT. The choice between the available drugs for HIT, namely lepirudin, danaparoid, and argatroban, has to be made according to the clinical presentation of the patient.     

Thrombin-activated thrombelastography for evaluation of thrombin interaction with thrombin inhibitors.

For intravenous anticoagulation, heparin has been the mainstay drug, but its use may be contraindicated in heparin-induced thrombocytopenia and thrombosis. Heparin alternatives including direct thrombin inhibitors are available, but clotting assays (e.g. partial thromboplastin time) measure the time required to form fibrin gel when only a small amount of thrombin is generated. It was hypothesized that the extent of thrombin inhibition varies among inhibitors, and thrombin-activated thrombelastography would provide useful data on therapeutic responses to thrombin inhibitors. Thrombin was added (0-100 nmol/l final concentration) to nonrecalcified whole blood to evaluate clot formation on thrombelastography. Effects of direct thrombin inhibitors (argatroban 3.75 microg/ml, bivalirudin 15 microg/ml, and lepirudin 3.0 microg/ml), and heparin cofactor II activator and dermatan disulfate (20 microg/ml) were evaluated in the presence of 100 nmol/l thrombin. The interactions of thrombin and respective inhibitors were also compared by fluorogenic thrombin substrate cleavage. Increasing concentrations of thrombin progressively shortened the lag time and increased viscoelasticity on thrombelastography. Only 20 nmol/l thrombin caused instantaneous clotting, but maximal viscoelastic force was obtained at 50-100 nmol/l thrombin. All thrombin inhibitors prolonged the lag time (lepirudin > bivalirudin > argatroban = dermatan disulfate), but full recovery of thrombelastography viscoelasticity was observed with argatroban and bivalirudin. Lepirudin abrogated clotting, and dermatan disulfate suppressed clot development on thrombelastography. Thrombin substrate cleavage was observed only for bivalirudin, and heparin cofactor II without dermatan disulfate. The modified thrombelastography technique using nonrecalcified whole blood may be useful in evaluating the extent and reversibility of thrombin blockade with direct or indirect thrombin inhibitors.     

Ecarin Clotting Time but not aPTT Correlates with PEG-Hirudin Plasma Activity

Background: Novel antithrombotic agents such as hirudin have shown promise in the therapy of acute coronary syndromes. PEG-hirudin (polyethyleneglycol conjugated hirudin) has been developed to provide a longer plasma half-life and more stable antithrombotic plasma levels. Privious trials indicated a narrow therapeutic window for hirudin and a number of aPTT (activated partial thromboplastin time)-monitored trials investigating hirudin in acute coronary syndromes had to be stopped because of intracranial bleeding complications. Objectives: The present study evaluates the ecarin clotting time (ECT), a parameter based on the conversion of prothrombin by the snake venom enzyme ecarin, for the monitoring of PEG-hirudin therapy. Methods: Plasma from either healthy volunteers (n=20) or from patients (n=10) suffering from unstable angina pectoris (UAP) was spiked with increasing PEG-hirudin concentrations. In a prospective randomized clinical trial patients with UAP were treated with intravenous PEG-hirudin or heparin over 72 hours. Patients were randomized to the following treatment groups: (1) heparin control group, n=15; (2) PEG-hirudin low dose (0.1[emsp4 ]mg/kg bolus, 0.01[emsp4 ]mg/kg/h infusion), n=19; (3) intermediate dose (0.15[emsp4 ]mg/kg and 0.015[emsp4 ]mg/kg/h), n=17; 4) high-dose (0.2[emsp4 ]mg/kg and 0.02[emsp4 ]mg/kg/h), n=16. Spiked plasma samples and plasma from UAP patients treated with i.v. PEG-hirudin were analyzed for aPTT, ECT, and PEG-hirudin levels. Results: A linear correlation up to the highest therapeutic concentrations could be observed between PEG-hirudin plasma concentrations and the ECT. This was true for both plasma samples spiked with PEG-hirudin in vitro as well as for samples taken from patients treated with i.v. PEG-hirudin (correlation coefficient 0.9, respect.) In contrast the aPTT did not show a reliable linear correlation to PEG-hirudin concentrations. Conclusion: Monitoring of PEG-hirudin therapy by ECT may help to avoid inadequate anticoagulation or overdosing. Thus, the safety and efficacy profile of PEG-hirudin therapy is likely to be enhanced by ECT monitoring.     

In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma

We investigated the activation of the nonenzymatic protein cofactors factor VIII and factor V in plasma when coagulation was initiated by thromboplastin. With sensitive bioassays, we were able to measure specifically the generation of activated factor VIII and activated factor V in plasma. Our results showed that when plasma was triggered with a relatively high concentration of thromboplastin, factor VIII and factor V were completely activated at the clotting time of plasma. However, when the generation of thrombin, but not that of factor Xa, was delayed by addition of hirudin to the plasma, factor Va was generated only at the time thrombin generation overcame the hirudin inhibition. In addition, generation of factor VIIIa correlated with thrombin generation and not with factor Xa generation. Furthermore, addition of large amounts of factor Xa to hirudinized plasma did not show detectable factor VIII or factor V activation. We concluded that in plasma activated with thromboplastin the enzyme responsible for activation of factor V and factor VIII is thrombin, not factor Xa.     

Tailoring haemostatic treatment to patient requirements – an update on monitoring haemostatic response using thrombelastography

Summary.  Currently, there is no single haemostasis laboratory test that has the capacity to accurately illustrate the clinical effects of procoagulant or anticoagulant interventions. Although the time course of thrombin generation in plasma and the endogenous thrombin potential (ETP) may be useful coagulation parameters, clotting involves components other than thrombin (e.g. platelets, fibrinogen). The continuous coagulation profiles of thrombelastography may provide a more accurate reflection of in vivo biology, covering initiation, development and final clot strength during whole blood clot formation. This method has helped to clarify the mechanism of action of whole blood clot formation, demonstrating the differences from clotting in plasma, and the importance of platelets and tissue factor titrations. It has also been used to investigate hypocoagulation (in haemophilia A, rare coagulation disorders, anticoagulant therapy and dilutional coagulopathy), hypercoagulation and the ex vivo testing of haemostatic interventions. Thrombelastography has been shown to reflect the clinical efficacy of activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) in patients with haemophilia A with inhibitors and in patients with acquired haemophilia. Overall, tailoring laboratory assays to illustrate and correlate with clinical phenotypes is essential for effective coagulation monitoring. Applying an algorithm of preoperative, perioperative and postoperative tests, including thrombelastography, may enable physicians to achieve this.     

Monitoring unfractionated heparin therapy: relationship between eight anti-Xa assays and a protamine titration assay.

Several studies have demonstrated that heparin assays, such as anti-activated factor X (anti-Xa) assays, can be successfully substituted for activated partial thromboplastin time for heparin dosage monitoring. A number of different assays are available and the relationship between results with different techniques is largely unknown. The aim of the present study was to assess the relationship between heparin assays by protamine titration and anti-Xa assays. Samples were collected from 43 patients receiving unfractionated heparin (UFH). In each sample, the heparin level was determined using a protamine titration assay and eight commercially available anti-Xa assays. The mean heparin level by protamine titration was 0.31 U/ml. Mean anti-Xa activity results ranged from 0.40 to 0.42 IU/ml for the three clotting-based assays, and from 0.32 to 0.40 IU/ml for five chromogenic assays. Thus mean results of different anti-Xa assays varied by up to 30%. The range of anti-Xa activity equivalent, on average, to 0.2-0.4 U/ml by protamine titration, considered to be the therapeutic range, was approximately 0.25-0.5 IU/ml, depending on the assay. The relationship between results of clotting and chromogenic methods was similar irrespective of whether or not warfarin-induced prolongation of international normalized ratios was present.     

Spontaneous antithrombin in a patient with benign paraprotein

A 66-year-old man with peptic ulcer disease developed a paraprotein that resulted in a spontaneously prolonged prothrombin time, activated partial thromboplastin time, and thrombin clotting time. Although the reptilase time was normal, the thrombin clotting time failed to correct with the addition of normal plasma, calcium, or protamine sulfate. The patient's purified fibrinogen was normal, but his serum contained an IgG that inhibited the clotting of normal plasma and purified fibrinogen in the presence of thrombin. In contrast to previously described paraproteins, this patient's IgG appeared to inhibit the activity of thrombin per se rather than to interfere with fibrinogen cleavage or fibrin polymerization. Although immunoprecipitation between thrombin and the paraprotein could not be demonstrated, the patient's purified IgG, in the presence of thrombin, decreased the thrombin activity on a chromogenic substrate. Further, increasing concentrations of thrombin overcame the inhibitory effect of the patient's paraprotein. Thus, the patient's paraprotein appeared to possess antithrombin activity.