Determination of the anti-F10a or anti-F2a generation action of rivaroxaban or dabigatran.

Dabigatran and rivaroxaban are new oral inhibitors of the thrombin (F2a) and F10a, respectively. Both inhibitors decrease the extrinsic or intrinsic generation of F10a/F2a in plasma. An innovative test for extrinsic F10a/F2a generation is the extrinsic coagulation activity assay (EXCA). With the EXCA the action of all F10a/F2a generation inhibitors can be quantified. The present work aimed to establish the mean therapeutic ranges of these two new drugs, as determined in the EXCA. Forty microlitres reconstituted lyophilized commercial pooled normal plasma, that had been supplemented with 0-0.48 mg/l dabigatran or rivaroxaban, in high-quality polystyrene U-wells (Brand781600), were incubated with 4 µl 1 ng/ml human placental tissue factor in 5% human albumin, 250 mmol/l CaCl2 (EXCA-trigger) for 1 min (EXCA-1) or 2 min (EXCA-2) at 37°C. 80 µl 2.5 mol/l arginine, 0.16% Triton X 100, pH 8.6, were added. After 3 min, 20 µl 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine was added and ΔA/t was determined (37°C). The mean therapeutic range (10-20% EXCA) of dabigatran is approximately 0.05-0.1 mg/l. The mean therapeutic range (10-20% EXCA) of rivaroxaban is approximately 0.13-0.22 mg/l. Mean prophylactic concentrations (20-40% EXCA) are approximately 0.02-0.05 mg/l dabigatran and approximately 0.05-0.13 mg/l rivaroxaban. These are the mean ranges. Individual plasma might well behave differently: either an individual patient could be more sensible or more resistant towards one or both of these drugs. It is suggested to perform EXCA tests for each individual patient to determine the real drug dosage he needs to reach 10-20% of normal EXCA for therapy or 20-40% of normal EXCA for prophylaxis.

Inhibition of thrombin generation in recalcified plasma.

Eight inhibitors of thrombin generation were compared in recalcified unfrozen plasma. Individual or pooled normal citrated plasma was supplemented on polystyrol flat-bottom wells (23 degrees C) with increasing concentrations of low-molecular-weight heparin, heparin, danaparoid, fondaparinux, hirudin, argatroban, corn trypsin inhibitor, or aprotinin. Thrombin was generated by addition of 5 microl fresh 250 mmol/l CaCl2 to 50 microl plasma in polystyrol flat-bottom wells and incubation for 20 min at 37 degrees C (recalcified coagulation activity assay). Arginine stopped hemostasis activation and then the generated thrombin activity was specifically quantified. The approximate 50% inhibitory concentrations of plasmatic anticoagulants for individual or pooled normal plasma are, respectively, 0.6 or 3.7 mIU/ml low-molecular-weight heparin, 0.3 or 1.6 mIU/ml heparin, 0.7 or 6.1 mU/ml danaparoid, 0.023 or 0.18 microg/ml fondaparinux, 75 or 230 pg/ml hirudin, 0.026 or 0.24 microg/ml argatroban, 1 or 2 U/ml corn trypsin inhibitor, and 2 or 4 KIU/ml aprotinin. The 50% inhibitory concentration values for corn trypsin inhibitor or aprotinin at plasmatic concentrations above 4-100 U/ml might increase pathologically the thrombin generation. The recalcified coagulation activity assay is a sensitive method to measure prothrombotic tendencies of blood or subtle concentrations of any plasmatic anticoagulant. It is suggested to analyze the individual patient's sensibility to certain plasmatic anticoagulants.

Determination of the global fibrinolytic state.

Fibrinolysis consists of a plasmatic part and a cellular part. A rapid global assay for plasmatic fibrinolysis is the fibrinolysis parameters assay (FIPA). Cellular fibrinolysis is measured by testing the clot lysis capacity using the microtitre plate clot lysis assay with polymorphonuclear neutrophils (CLA-PMN). Individual citrated plasma or pooled normal plasma (50 microl) of 232 patients was recalcified, incubated for 90 min at 37 degrees C, oxidized with 0 or 1.5 mmol/l (final concentration) chloramine-T, and supplemented with 50 microl respective polymorphonuclear neutrophil plasma. The turbidity of the clots was measured at 405 nm after 12 h and 60 h (37 degrees C). Plasma (50 microl) was also incubated with 5 microl of 100 IU/ml urokinase, 6 mmol/l tranexamic acid, 6% human albumin for 10 min (37 degrees C). Then 100 microl of 0.5 mmol/l Val-Leu-Lys-pNA in 2.45 mol/l arginine, pH 8.6, was added and the increase in absorbance with time was measured. The different CLA-PMN assay versions correlated with each other with r = 0.543-0.782. Cellular fibrinolysis (34 +/- 30% lysis; normal: 25 +/- 10%) did not correlate with the FIPA (72 +/- 27%; normal: 100 +/- 15%), prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, or the blood counts of thrombocytes, leukocytes, or polymorphonuclear neutrophils. Chloramine (1.5 mmol/l) oxidation of the microclots favours their fibrinolytic breakdown, especially if lysis-resistant microclots are oxidized. The FIPA and CLA-PMN are new economical tests for the fibrinolytic state in patient blood.

The efficiency of anti-activated factor X and anti-activated factor II anticoagulants.

Six thrombin-generation inhibitors or thrombin inhibitors were compared in the extrinsic coagulation activity assay (EXCA), where the normal thrombin generation is about 1 IU/ml within 1 min (37 degrees C). Unfrozen pooled normal citrated plasma was supplemented on flat-bottom wells (23 degrees C) with increasing concentrations of dalteparin, danaparoid, heparin, fondaparinux, hirudin, or argatroban. To 50 microl plasma, 5 microl of 1.5 ng/ml tissue factor, 6% bovine serum albumin, and 250 mmol/l CaCl2 were added. After 1 and 2 min coagulation reaction time at 37 degrees C (EXCA-1 and EXCA-2), 100 microl of 2.5 mol/l arginine and 0.16% Triton X 100, pH 8.6, were added. After 3 min (23 degrees C), 25 microl of 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, were added, and the linear increase in absorbance with time was determined at 405 nm. The 50% inhibitory concentrations of plasmatic anticoagulants tested in the EXCA-1 (37 degrees C) were 0.025 IU/ml dalteparin, 0.13 U/ml danaparoid, 0.12 IU/ml heparin, 1.3 microg/ml fondaparinux, 2.4 ng/ml hirudin, and 1 microg/ml argatroban. From the 50% inhibitory concentration of hirudin it can be concluded that inhibition of about 30 mIU/ml thrombin halves the normal EXCA-1 value (i.e. if about 0.1 IU/ml thrombin are inactivated, then thrombin cannot self-amplify its generation 10-fold). The efficiency of any clinically used plasmatic anticoagulant can be monitored in the EXCA.

Influence of coagulation factors on extrinsic thrombin generation.

The extrinsic coagulation activity assay (EXCA) is a new thrombin generation test for the tissue factor pathway of coagulation. The EXCA was performed with 10 parts citrated plasma of different contents of fibrinogen. One part tissue factor, 250 mmol/l CaCl(2), generating about 1 IU/ml thrombin within 1 min (37 degrees C). After 0-30 min 2.5 mol/l arginine (pH 8.6) Generated thrombin was detected by addition of CHG-Ala-Arg-pNA and measurement of triangle upA/t. The EXCA is dependent on factors 10% of the factor VII norm in the sample achieves 70-80% of the thrombin generation norm. The EXCA is not dependent on factors VIII, IX, XI and XII. Even in antithrombin III-deficient plasma, a phase of thrombin inhibition appears after the thrombin peak. Supplemented purified fibrinogen resulted in decreased thrombin generation in the important. Fibrinogen seems to act as antithrombin I; thrombin might be entrapped in the nascent fibrin. The EXCA is suitable to diagnose the level of extrinsic factors in patient plasma.

Thrombin generation by hemolysis.

Hemolysis is the fragmentation of erythrocytes into microparticles (Hb-MP). Clinical hemolysis can result in a severe procoagulant state. The influence of Hb-MP on thrombin generation was quantified. Unfrozen citrated normal plasma (five donors) was supplemented with 0 or 1 g/l Hb-MP obtained through erythrocyte destruction by hypotonic lysis, freezing/thawing, or blood oxidation with 1 or 2 mmol/l chloramine-T. Pooled normal plasma was supplemented with 0-10 g/l Hb-MP and with 0-1 IU/ml low-molecular-weight heparin (dalteparin). Samples (50 microl) were tested in the recalcified coagulation activity assay. At 10 min coagulation reaction time the hypotonic lysis of erythrocytes appears to be the most procoagulant condition, followed by twice freezing/thawing, three times freezing/thawing, and once freezing/thawing. Oxidation of whole blood with 1 or 2 mmol/l chloramine-T decreases thrombin generation by about 20 or 50%, respectively. The thrombin generation in 1 mmol/l chloramine-T or 2 mmol/l oxidized plasma decreases by about 70 or 85%, respectively. The 50% inhibitory concentrations of low-molecular-weight heparin against recalcified thrombin generation are 0.01, 0.025, or 0.035 IU/ml for plasma supplemented with 0, 0.1, or 1 g/l Hb-MP, respectively. The recalcified coagulation activity assay allows one to quantify thrombin generation in critical hemolytic samples. It is suggested to find the appropriate pharmacologic dose of low-molecular-weight heparin.

Inhibition of extrinsic hemostasis activation by low-molecular-weight heparin.

Low-molecular-weight heparins (LMWHs) are very important drugs; unfortunately, the routine global hemostasis assays activated partial thromboplastin time and prothrombin time are not sensitive to LMWHs. Here the 50% inhibitory concentration (IC(50)) values of heparin and LMWHs on extrinsic thrombin generation are determined. Pooled normal plasma was supplemented with 0-2 IU/ml unfractionated heparin, 0-2 IU/ml LMWH dalteparin, or 0-20 microg/ml pentosanpolysulfate in 5-ml polystyrole tubes (23 degrees C) and tested in the tissue-factor-triggered extrinsic coagulation activity assay (EXCA): 50 microl plasma + 5 microl tissue factor/CaCl(2), 1 and 2 min incubation time at 37 degrees C (coagulation reaction time for EXCA-1 and EXCA-2); + 100 microl of 2.5 mol/l arginine (pH 8.6), 20 min at room temperature; + 50 microl of 1 mmol/l CHG-Ala-Arg-pNA, 1.25 mol/l arginine; increase in absorbance/time at 23 degrees C; calibrator = 1 IU/ml bovine thrombin in 6.7% human albumin replacing the plasma sample; in EXCA-1, about 1 IU/ml thrombin is generated in pooled unfrozen normal citrated plasma. The IC(50) values in EXCA-1 are 0.1 IU/ml heparin, 0.02 IU/ml LMWH, and 4.7 microg/ml pentosanpolysulfate. In ECXA-2 the IC(50) values are 0.07 IU/ml, 0.01 IU/ml, and 4.6 microg/ml, respectively. The EXCA reflects the efficiency of anticoagulants on plasmatic coagulation. It is suggested to adjust the dosage of LMWH according to the EXCA value; about 30% of normal extrinsic thrombin generation might be the correct dose for prophylactic anticoagulation.