Effects of unfractionated and low-molecular-weight heparins on fibrin polymerization

In vitro experiments show that therapeutic doses of unfractionated and low-molecular-weight heparins inhibit fibrin polymerization. Sodium and calcium salts of unfractionated heparins exhibit antipolymerization activity. The activity of low-molecular-weight heparins is higher, at 0.75 anti-X_a units/ml fraxiparin produces a stronger inhibiting effect on polymerization of fibrin monomers than clivarin. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 9, pp. 346–347, September, 1997     

Ristocetin and the thrombin clotting time.

The addition of the antibiotic ristocetin to plasma accelerated the thrombin clotting time (TCT) in 20 out of 22 subjects. Prior incubation of ristocetin with thrombin or plasma did not alter its effect on the TCT. Ristocetin accelerated clotting greatly at low but not at high levels of thrombin. A simple linear correlation between heparin concentrations and the TCT was demonstrated when ristocetin at 2.5 mg per ml was added to plasma containing between 0.05 and 0.5 unit of heparin per ml. There are implications for assay procedures involving heparin and the TCT.     

Follow-up of the treatment by direct thrombin inhibitors: activated partial thromboplastin time or ecarin clotting time

The clinical use of the direct inhibitors of thrombin requires a reliable test to monitor the treatment and to predict the hemorragic risk. The activated partial thromboplastin time (APTT) is the most common test used to monitor treatment with unfractionated heparin. Thus APTT has been first chosen to follow patients treated with direct thrombin inhibitors, but studies have shown that it was probably not the most appropriate test. Indeed, APTT values were not well correlated with the dose administered and were dependent on the type of the thrombin inhibitor used and on the APTT reagent. The ecarin clotting time (ECT), which converts prothrombin into meizothrombin has been then tested and seemed to be a better test. In vitro studies have shown a good correlation between ECT and the different concentrations of thrombin inhibitors. Furthermore, the ECT in contrast to APTT is not sensitive to heparin or oral anticoagulant and interindividual variations are low with ECT. ECT which is a reliable test and is easy to perform seems to be a more appropriate test to monitor treatment with direct thrombin inhibitors but further studies are needed to validate its use in a clinical setting.     

Effects of unfractionated heparin, low-molecular-weight heparin, and heparinoid on thromboelastographic assay of blood coagulation

Thromboelastography (TEG) has been used increasingly as an intraoperative hemostasis monitoring device. Low-molecular-weight heparins are given increasingly to reduce the development of antibodies against the heparin-platelet factor 4 complex, and heparinoids are given to patients who have developed the antibody. We studied the effect of unfractionated heparin, a low-molecular-weight heparin (enoxaparin sodium [Lovenox]), and a heparinoid (danaparoid sodium [Orgaran]) on blood clotting assayed with TEG (TEG clotting) in vitro and the efficacy of protamine sulfate and heparinase for reversing the effect. Heparin, enoxaparin, and danaparoid all caused a dose-dependent inhibition of TEG clotting of normal blood. Concentrations of enoxaparin and danaparoid that totally inhibited TEG clotting only minimally prolonged the activated partial thromboplastin time. While inhibition of TEG clotting by heparin and enoxaparin was reversed by protamine sulfate and heparinase, inhibition by danaparoid was reversed only by heparinase. Abnormal TEG clotting was observed in patients receiving enoxaparin whose plasma level of the drug was more than 0.1 antiXa U/mL. However, the degree of TEG abnormality did not always coincide with plasma levels of the drug.     

The thrombin clotting time. Evaluation of Thromboquik, a commercial thrombin reagent, and observations on sensitivity and on centrifugation

The thrombin clotting time (TCT) is an important screening test of coagulation, but thrombin can be a troublesome reagent to use, and in general the TCT has not been well standardized, even with respect to the ideal normal range. Thromboquik is a thrombin reagent that is convenient in its day-to-day use and that exhibits prolonged stability after reconstitution. As an extension of an evaluation of this commercial thrombin reagent, the authors have made several observations about the TCT itself. In a comparison between multiple pairs of TCT reagents, the relative extent of departure of clotting times from normal, for any given abnormal plasma sample, was not predictably related to the mean normal clotting times of the two reagents. The authors also concluded that centrifugation of blood at 1,500 X g is inadequate to prepare plasma for the TCT.     

Migration inhibition factor and the blood clotting system: effects of defibrination, heparin and thrombin.

Heparin is not suitable as an anticoagulant in the leucocyte migration test used to demonostrate the presence of migration inhibition factor (MIF) due to a rapid disappearance of the response after even a short storage of the blood. The use of defibrinated blood is highly recommended and defibrinated blood can be stored for at least 90 min without any diminution of the response. The change in response when using heparinized blood is not due to any direct effect of heparin, because heparin has no effect when added to defibrinated blood. However, heparin, added together with thrombin, is capable of abolishing the MIF effect completely. The basis for this phenomenon is most probably the binding of the heparin-antithrombin cofactor (AT III) to a complex with heparin and thrombin. The activity of MIF requires the presence of AT III, its esterase-inhibiting activity probably being crucial, in order to express MIF activity on macrophages. This mechanism forms a link between certain cellular immune reactions and the blood-clotting system.     

Presence of direct thrombin inhibitors can affect the results and interpretation of lupus anticoagulant testing

In vitro experiments were conducted to determine whether the direct thrombin inhibitors argatroban and lepirudin can interfere with the results of lupus anticoagulant (LA) testing. Concentration-response curves were generated to calculate the concentration of anticoagulant that prolongs the activated partial thromboplastin time (aPTT) to 75 seconds (2.5 times the baseline average). Corresponding concentrations of anticoagulant were added to plasma samples before running dilute Russell viper venom tests (DRVVTs) and LA-sensitive aPTTs (PTT-LAs). Because the DRVVT test system contains an antiheparin agent, DRVVT results were not prolonged in the presence of heparin. With argatroban added to normal plasma samples, neither the DRVVT percent correction of ratio nor the DRVVT test/confirm ratio were elevated, but when added to LA-positive plasma, some false-negative results were observed. Lepirudin increased the DRVVT percent correction of ratio and the DRVVT test/confirm ratio into a range that could lead to false-positive identifications of LAs. In sharp contrast to the DRVVT test system, distinction between LA-positive and LA-negative plasma samples was maintained and possibly even enhanced in the platelet neutralization procedure correction phase of the PTT-LA test system.     

Modified plasma-based ecarin clotting time assay for monitoring of recombinant hirudin during cardiac surgery

Recombinant hirudin (r-hirudin) is being used increasingly for therapeutic anticoagulation in patients with heparin-induced thrombocytopenia undergoing cardiovascular surgery. Although multiple laboratory methods are available for measuring r-hirudin, the ecarin clotting time (ECT) is the most commonly used for this purpose. Ecarin (extracted from snake venom) converts prothrombin to meizothrombin, which promotes clot formation. Direct thrombin inhibitors, like r-hirudin, bind meizothrombin and yield a linear, dose-dependent prolongation of ECT. Low levels of prothrombin and fibrinogen in plasma samples can lead to higher ECT; suggesting falsely elevated r-hirudin levels. A modified ECT assay with prothrombin and fibrinogen in excess was optimized using an orthogonal array method to eliminate the variations in patients' plasma prothrombin and/or fibrinogen levels for accurate determinations of plasma r-hirudin levels. By using the modified ECT assay, falsely elevated r-hirudin levels can be avoided in patients undergoing cardiopulmonary bypass, thus providing reliable and accurate r-hirudin monitoring in this clinical setting.     

Effect of direct thrombin inhibitors, bivalirudin, lepirudin, and argatroban, on prothrombin time and INR values

Direct thrombin inhibitors (DTIs) represent a new class of promising anticoagulation agents. The DTIs frequently are used to provide initial anticoagulation, with long-term therapy requiring eventual transition to coumarins. Unfortunately, DTIs not only prolong the activated partial thromboplastin time but also can affect international normalized ratio (INR) values. We approximated the DTI effect on INRs by each drug to pooled plasma at concentrations between 0.1 and 1.2 microg/mL. We then concurrently tested these samples using 14 prothrombin time (PT) reagents. By using repeated measures analysis of variance, we found significant differences (P < .05) between the median INRs for lepirudin and argatroban for all PT reagents, between lepirudin and bivalirudin for all reagents except PT-Fibrinogen HS Plus (P = .07), and between bivalirudin and argatroban for all reagents except Thromborel S (P = .05). The DTI effect on INRs was dependent on drug, drug concentration, and reagent. Argatroban had the most effect on INRs, while lepirudin had the least effect. Reagents with a lower international sensitivity index were less affected by DTI; ThromboMax HS was the least sensitive PT reagent to any DTI.     

Simultaneous determination of precursors of kallikrein,plasmin, and thrombin and their inhibitors in human blood plasma

Prekallikrein, plasminogen, and prothrombin of human blood plasma are separately activated by kaolin, streptokinase, or thromboplastin. Measurement of the TAME-esterase (TAME: N-tosyl-L-arginine methyl ester) activity of each of the enzymes thus produced and its changes during incubation of the plasma with the activator can be used to estimate the content of kallikrein, plasmin, and thrombin and their inhibitors. Evidence is given that under the conditions of determination as described activation is specific for each enzyme and does not affect the level of the other precursors. The method is developed in modifications enabling the values of seven indices to be obtained with 0.4–0.7 ml of blood plasma.Insitute of General Pathology and Pathophysiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 632–634, May, 1976.     

Ecarin chromogenic assay--a new method for quantitative determination of direct thrombin inhibitors like hirudin

A new sensitive and precise method for quantitative determination of direct thrombin inhibitors is described, the ecarin chromogenic assay (ECA). Ecarin is used as the specific prothrombin-activating principle. The cleavage of a chromogenic substrate by meizothrombin is inhibited in a concentration-dependent fashion by direct thrombin inhibitors. For the ECA, the linear measuring range is about 0.1-3.0 microg hirudin/ml plasma. Coefficients of variations between 2.3 and 4% over the whole concentration range were achieved. The ECA has proved to be more sensitive than the compared tests (ecarin clotting time and a thrombin-based chromogenic assay); a detection limit of 0.011 microg hirudin/ml and a quantitation limit of 0.032 microg hirudin/ml were calculated. The ECA is independent of the variations of the coagulation variables fibrinogen and prothrombin. Neither heparin nor oral anticoagulants interfere with the ECA.     

Comparison of an immunochemical assay for plasma fibrinogen and a turbidimetric thrombin clotting technique to discriminate hyperlipidaemic patients from healthy controls.

Plasma samples from patients attending a lipid clinic (n = 14) and healthy control subjects (n = 21) were assayed for fibrinogen using an immunochemical method (radial immunodiffusion) and a turbidimetric assay based on the thrombin clotting technique. The patients had significantly higher plasma fibrinogen concentrations than controls by both methods, but there was significant overlap between the two groups when fibrinogen was assayed by the thrombin clotting technique; there was almost complete separation of the two groups using the immunochemical assay. This difference in overlap could not be attributed to the presence or absence of fibrinogen degradation products. These findings may have important implications for the choice of method for determining plasma fibrinogen when assays are used for the assessment of cardiovascular risk. It is recommended that plasma fibrinogen should be assayed by both an immuno-chemical and a thrombin clotting method.