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PubMed2004

Comparing direct thrombin inhibitors using aPTT, ecarin clotting times, and thrombin inhibitor management testing.

BACKGROUND: Patients with heparin-induced thrombocytopenia and thrombosis may be acutely anticoagulated with direct thrombin inhibitors (DTIs). The anticoagulation is typically monitored using the activated partial thromboplastin time (aPTT) or ecarin clotting time (ECT). OBJECTIVE: To compare 14 methods for measuring aPTT, as well as ECT and thrombin inhibitor management test (TIM), in samples containing DTIs. METHODS: DTIs were added to pooled normal plasma to achieve low (0.1-1.2 microg/mL) and high (1.5-8.0 microg/mL) drug concentrations. Each low-concentration DTI sample was tested using all aPTT reagents, while each low- and high-concentration DTI was tested using the ECT and TIM. RESULTS: All aPTT reagents had a significant dose-dependent correlation with drug concentration. Only Actin FSL and APTT-S demonstrated equivalent aPTT ratios obtained from any DTI. The TAS-aPTT was the most sensitive aPTT reagent to argatroban, with the aPTT ranging from 52.7 to 121.2 seconds corresponding to 0.1 to 1.2 microg/mL of drug concentration. The TAS-aPTT and Pathromtin were the most sensitive aPTT reagents to bivalirudin, with aPTTs of 87.4 seconds and 101.5 seconds, respectively, at 1.2 microg/mL of drug. Pathromtin was the most sensitive aPTT reagent to lepirudin, with a maximum aPTT of 108.9 seconds at 1.2 microg/mL of drug. There was no statistically significant difference between the TIM and ECT clotting times for each DTI. Lepirudin and bivalirudin ECT and TIM clotting times were equivalent. CONCLUSIONS: There are unique differences between reagent manufacturers in the monitoring of DTIs. Acceptable alternatives to aPTT monitoring of DTI anticoagulation include the ECT and TIM.

Arginine↗
PubMed1992

[Interaction of human thrombin I fragment, prethrombin I, and alpha-thrombin with tissue thromboplastin].

The binding of 125I-labeled prothrombin fragment I. prethrombin I and alpha-thrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 of EDTA was studied. The experimental curves plotted in the Scatchard coordinates testify to the presence in thromboplastin of two types of fragment I binding sites: those with a high (Kd = 7.6 x 10(-6) M) and moderate (Kd = 1.3 x 10(-8) M) binding affinity. The parameters of fragment I binding and their changes reproduced, for the most part, the mode of prothrombin binding observed in previous studies. The experimental results provide indirect evidence in favour of a hydrophobic role of Ca(2+)-dependent binding of prothrombin fragment I to thromboplastin. The binding of prethrombin I was nonspecific and Ca(2+)-independent, whereas alpha-thrombin showed a relatively high level of nonspecific electrostatic binding which was competitively inhibited by Ca2+. Thromboplastin proteins interacted (both directly and in a Ca(2+)-independent fashion) with all the prothrombin derivatives under study.

Binding Sites↗
PubMed1989

Anti-thrombin activity in cultured aortic and capillary endothelial cells: binding, internalization and degradation of thrombin.

Thrombin (Th) binds specifically to confluent cultures of adult bovine aortic (ABAE) and bovine brain capillary (BBC) endothelial cells. Saturation of 125I-Th binding is observed after 1 h exposure to the ligand and at an extracellular concentration of 0.5 and 1.0 microgram/ml for ABAE and BBC cells, respectively. Under optimal conditions both ABAE and BBC cultures bind about 2 to 5 ng/10(6) cells, which represents about 20% of Th binding to bovine corneal endothelial (BCE) cells. Under optimal conditions less than 30% of the total cell associated 125I-Th is internalized in ABAE and BBC cells, while in BCE cells the extent of internalization is more than 50%. The internalized 125I-Th is degraded both in ABAE and BBC cells as previously demonstrated in BCE cells. As analyzed by SDS-PAGE, 17%, 22% and 77% of the bound 125I-Th is in complex with anti-thrombins (anti-Ths) in BBC, ABAE and BCE cultures, respectively. ABAE cells possess 3 types of complexes, one which appears only on the cell surface with a molecular weight of 78 kDa, and two others which appear only in the conditioned medium (CM) with molecular weights of 84 and 85 kDa. BBC and BCE cells demonstrate only one type of complex with a molecular weight of 77 kDa which appears both on the cell surface and in the CM. The 125I-Th 77 kDa complex formed in the CM of BCE cells is recognized and bound by BBC cells and ABAE cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Activation-induced exposure of the thrombin anion-binding exosite. Interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite-specific antibody.

The activation of serine protease zymogens involves conformational changes that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.

Amino Acid Sequence↗
PubMed1997

Interaction of a thrombin inhibitor and a platelet GP IIb/IIIa antagonist in vivo: evidence that thrombin mediates platelet aggregation and subsequent thromboxane A2 formation during coronary thrombolysis.

We examined the effect of a specific thrombin inhibitor, Ro 46-6240, alone and combined with an antagonist of the platelet GP IIb/IIIa, Ro44-9883, on the response to tissue-type plasminogen activator in a canine model of thrombolysis. Platelet activity was determined by measuring the excretion of 2,3-dinorthromboxane (TX)B2, an enzymatic metabolite of TXA2. Ro 46-6240 administered before tissue-type plasminogen activator induced a dose-dependent prolongation of the activated partial thromboplastin time and prothrombin time. The time to reperfusion decreased dose-dependently (P < .01) to 10 +/- 6 min vs. 52 +/- 5 min in controls. Ro 46-6240 also prevented reocclusion, which occurred in every case in control experiments. Urinary excretion of 2,3-dinor-TXB2 increased from 3 +/- 1 to 37 +/- 9 ng/mg creatinine in controls after reperfusion. This increase was reduced in a dose-dependent fashion by Ro 46-6240, such that at the highest dose, urinary 2,3-dinor-TXB2 after reperfusion was 5.6 +/- 1 ng/mg creatinine. Similar functional and biochemical effects were seen when a subthreshold dose of Ro 46-6240 was combined with Ro 44-9883. At the dose used, Ro 44-9883 alone abolished platelet aggregation ex vivo but failed to modify the response to tissue-type plasminogen activator or the excretion of 2,3-dinor-TXB2 after reperfusion (51 +/- 6 ng/mg creatinine, n = 3). However, the combination of Ro 44-9883 and Ro 46-6240 reduced the time to reperfusion (40 +/- 8 vs. 68 +/- 15 min; n = 7, P < .05), prevented reocclusion and abolished the rise in urinary 2,3-dinor-TXB2 (5 +/- 1 ng/mg creatinine, n = 4). These findings suggest that thrombin mediates platelet activation during coronary thrombolysis. The increased platelet activity results in platelet aggregation and a subsequent increase in TXA2 formation.

Acetates↗
PubMed1997

Thrombin-binding properties of thrombin aptamer derivatives.

We synthesized a single-stranded DNA with triple G-quartet structure and found that its thrombin-binding activity at 37 degrees C is as much as that of thrombin aptamer with double G-quartet structure which is a product of in vitro selection and amplification. This result suggests that the sequence motifs obtained by in vitro selection and amplification method might not be the only answer.

Base Sequence↗
PubMed1997

Thrombin Hypothesis: The TIMI 9B and GUSTO IIB Trials Have Successfully Disproven/Proven the Thrombin Hypothesis.

The direct thrombin inhibitor, hirudin, was tested in two trials. The TIMI 9B trial randomized patients receiving thrombolytic therapy for acute myocardial infarction to receive hirudin or heparin. The GUSTO IIB trial randomized patients with or without electrocardiographic ST-segment elevation (i.e. thrombolytic- and non-thrombolytic-eligible patients). In the combined trials at 30 days there was a non-significant 14% reduction in myocardial infarction, but no effect on mortality. There are a number of factors in these two trials, including dose selection, timing of administration and duration of drug therapy, that may have led to an underestimate of the potential benefits of hirudin. Further trials are therefore required to test the thrombin hypothesis.

Journal Article↗
PubMed1997

A low molecular weight, selective thrombin inhibitor, inogatran, vs heparin, in unstable coronary artery disease in 1209 patients. A double-blind, randomized, dose-finding study. Thrombin inhibition in Myocardial Ischaemia (TRIM) study group.

BACKGROUND: Unstable coronary artery disease, i.e. unstable angina or non-Q wave myocardial infarction, is caused by rupture of atheromatous plaques initiating thrombus formation. Thrombin is thought to be pivotal in platelet activation and thrombus growth. The aim of the present study was to compare the effect of three different doses of a novel, low molecular weight, selective thrombin inhibitor (inogatran) with that of heparin in unstable coronary artery disease. The composite primary end-point was death, incidence of myocardial infarction, refractory angina or recurrent angina after 7 days. Secondary end-points were the same after 3 and 30 days, as were death and myocardial infarction with or without refractory angina on all three occasions. METHODS: Patients (n = 1209) admitted with suspected unstable angina, or non-Q wave myocardial infarction, were randomly assigned to double-blind treatment with inogatran or heparin bolus doses. This was followed by a 3 day infusion with a low (LDI), medium (MDI), or high (HDI) dose of inogatran, aiming at plasma concentrations of 0.15, 0.4 or 0.8 micromol.l-1, or heparin initiated at 1200 U.h-1. FINDINGS: Treatment with inogatran resulted in median activated partial thromboplastin times after 24 h of 36, 44 and 53 s in the low, medium and high dose inogatran groups, respectively, as compared to 54 s in the heparin group (ns). At the end of the infusion, after 3 days, heparin-treated patients had fewer composite events (29.5%) than inogatran-treated patients (LDI = 39.4%, MDI = 37.6%, HDI = 36.1%; P = 0.01). The event rate after 7 days with respect to the primary end-point, however, did not differ between the groups (heparin = 41.0%, LDI = 45.7% MDI = 45.9%, HDI = 45.5%; ns). Death and myocardial infarction occurred less frequently in the heparin group (0.7%) than in the three inogatran groups (LDI = 3.6%, MDI = 2.0%, HDI = 4.0%; P < 0.05) after 3 days. After 7 days, this difference was attenuated (heparin = 2.6% vs LDI = 4.0%, MDI = 4.3%, HDI = 6.7%; P = 0.10). After 30 days there were no significant differences in event rates between the four groups. Major bleeding occurred in 1.1% of the patients within 7 days, with no differences between the groups. CONCLUSIONS: During study drug infusion the event rate was very low in the heparin group, and none of the inogatran dosages were better than heparin in preventing ischaemic events. Furthermore, there was no relationship between event rate and inogatran dosage. Thus, this study does not indicate that the efficacy of inogatran would improve with higher doses, despite a clear dose-effect as regards the prolongation of activated partial thromboplastin time. After cessation of heparin or inogatran there was an increase in event rates, suggestive of a rebound phenomenon.

Aged↗
PubMedDate not supplied

Influence of heparin, protamine and polybrene on the time integral of thrombin generation (endogenous thrombin potential).

We investigated the anticoagulating and heparin-neutralizing properties of protamine and polybrene (hexadimethrine bromide), using the endogenous thrombin potential (ETP) as the parameter to access plasma coagulability. The hypocoagulability induced by high doses of heparin (3 IU/ml) could be reversed by addition of protamine to a very limited extent only. Polybrene on the other hand did neutralize heparin at the equivalent concentration and a two-fold excess did not influence the ETP parameters. In vivo neutralization of high-dose heparin with protamine should therefore be reconsidered. Our experiments suggest polybrene to be superior over protamine with respect to neutralization of high doses of heparin.

Anticoagulants↗
PubMed1989

Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Heparin versus recombinant hirudin, a specific thrombin inhibitor.

The effect of recombinant hirudin and the dosage of heparin on acute platelet-thrombus deposition during carotid angioplasty in anesthetized pigs was prospectively assessed. Fifty-five animals (mean weight, 33.9 kg) were randomized to one of six heparin dosages: heparin boluses of 35, 50, 100, 150, 200, or 250 units/kg followed by a continuous infusion of 35, 50, 100, 150, 200, or 250 units/kg/hr, respectively. Another five pigs received a bolus of 1 mg/kg hirudin (recombinant desulphatohirudin), a specific thrombin inhibitor, followed by an infusion of 1 mg/kg/hr. Bilateral carotid angioplasty was performed in all pigs 20 minutes after starting the infusion; they were sacrificed 57 +/- 12 minutes after the procedure. Deep medial arterial injury was present in approximately 75% of the dilated segments, and subendothelial injury in the remaining 25%. Mean log of number of platelets and molecules of fibrinogen per centimeter squared of deep injury in segments from all the animals treated with heparin were 4.74 +/- 1.03 and 5.02 +/- 0.64, respectively. A regression analysis showed an inverse correlation of the log of platelet deposition with the heparin group (r = -0.56, p = 0.0001) with administered total units of heparin (r = -0.55, p = 0.0003) and with mean plasma heparin concentration (r = -0.55, p = 0.0004) in deeply injured arteries. Similar inverse relations were obtained for fibrinogen. In contrast, the deposition of platelets and fibrinogen in subendothelial injury was very low and independent of the heparin administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Angioplasty, Balloon↗
PubMed2002

A dose-ranging study of the oral direct thrombin inhibitor, ximelagatran, and its subcutaneous form, melagatran, compared with dalteparin in the prophylaxis of thromboembolism after hip or knee replacement: METHRO I. MElagatran for THRombin inhibition in Orthopaedic surgery.

The novel, oral direct thrombin inhibitor, ximelagatran (formerly H 376/95), represents an advance in antithrombotic therapy through its oral availability. After oral administration, ximelagatran is converted to its active form, melagatran. Melagatran can also be administered subcutaneously (s.c.). The results from the first clinical study with ximelagatran are reported. In this randomized, parallel-group, controlled study, 103 patients scheduled for elective total hip or total knee replacement received s.c. melagatran (1, 2 or 4 mg bid) for 2 days commencing immediately before surgery, followed by oral ximelagatran (6, 12 or 24 mg bid) for 6-9 days. Another 33 patients received dalteparin 5000 IU s.c. once daily, started the evening before surgery, for 8-11 days. At bilateral venography, deep vein thrombosis was found in 20.5% (16/78) of patients who had received s.c. melagatran and oral ximelagatran and in 18.5% (5/27) of patients in the dalteparin group. The study did not evaluate a dose-response for efficacy, and no differences between the three dose levels of melagatran and ximelagatran were shown. No pulmonary embolism was diagnosed during treatment. Total bleeding in the s.c. melagatran plus oral ximelagatran groups showed no dose-response and was similar to that seen in the dalteparin group. The pharmacokinetic properties of melagatran in the surgery patients were consistent with those observed for healthy subjects, and the APTT ratio, which increased non-linearly with plasma melagatran concentration, showed a consistent concentration-effect relationship during the treatment period. Ximelagatran and melagatran were well tolerated. In conclusion, ximelagatran and its active form melagatran appear to be promising agents for the prevention of venous thromboembolism following orthopaedic surgery.

Administration, Oral↗
PubMed2003

Thrombin generation assessed as endogenous thrombin potential in patients with hyper- or hypo-coagulability.

BACKGROUND AND OBJECTIVES: The endogenous thrombin potential (ETP) represents the balance between pro- and anti-coagulant forces operating in plasma and can be used to investigate hyper- and hypo-coagulability. As a preliminary step to larger clinical studies we investigated the effect on ETP of phospholipids, tissue factor (TF) and residual platelets in frozen plasma. DESIGN AND METHODS: We investigated platelet-poor and platelet-rich plasmas from healthy subjects, patients on oral anticoagulants (OA), or with hemophilia and women on oral contraceptives (OC), chosen as examples of the normal, hypo- and hyper-coagulable states in which ETP has been reported to be impaired. RESULTS: Phospholipids had only a slight effect on ETP in all conditions except in women on OC, in whom the best diagnostic efficacy was observed at 0.5 microM. TF had only a slight effect in all conditions except hemophilia, in which an ETP impairment was observed only at low (1 pM) concentration. Residual platelets had considerable effects on ETP in frozen plasmas, but this was abrogated by filtration before freezing. ETP in platelet-rich plasma at 150x103/mm3 was similar to that obtained in filtered-plasma with 1.5 microM phospholipids in healthy subjects, patients on OA and patients with severe hemophilia, but not in those with mild- or moderate-hemophilia, where the ETP was higher in platelet-rich plasma. INTERPRETATION AND CONCLUSIONS: The results suggest that the method can be used for investigations on the clinical value of ETP. Platelet-rich and platelet-poor plasma are suitable testing materials. The latter should be filtered before freezing to minimize the effect of residual platelets.

Anticoagulants↗
PubMed1993

Recombinant human soluble thrombomodulin delivers bounded thrombin to antithrombin III: thrombomodulin associates with free thrombin and is recycled to activate protein c.

Recombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (Kd = 2.6 nM) or PC activation (Kd = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (Kd < 0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Animals↗
PubMed1986

Management of anti-thrombin III deficiency during pregnancy without administration of anti-thrombin III.

We report a patient with hereditary antithrombin III deficiency who was successfully treated with heparin throughout pregnancy. Functional antithrombin III levels fell to 0.32 U/ml during heparin treatment, but it was possible to achieve a heparin effect, measured by the activated partial thromboplastin time, thrombin clotting time and heparin assay with subcutaneous heparin in doses of 30,000 U to 35,000 U/24 hours. This achieve an long term heparin effect was obtained without the need for antithrombin III infusions.

Adolescent↗