Antithrombin-heparin Covalent Complex Research Articles

Inhibition of plasmin generation in plasma by heparin, low molecular weight heparin, and a covalent antithrombin-heparin complex.

: The clinical limitations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) led to the development of an antithrombin-heparin covalent complex (ATH), which displays superior anticoagulant abilities compared with UFH. A recent study investigating its interaction with fibrinolysis showed that ATH inhibited free and fibrin bound plasmin and decreased plasmin generation on fibrin clots. These studies were conducted using purified components and did not elucidate the interaction of ATH with plasmin in the presence of its natural inhibitors α2-antiplasmin (α2-AP) and α2-macroglobulin (α2-M). The aim of this study was to determine the effects of ATH, UFH, and LMWH on plasmin generation in plasma, under more physiological conditions. Plasmin generation in plasma in the absence and presence of anticoagulants was initiated by tissue plasminogen activator and soluble fibrin fragments, and plasmin and plasmin-α2-M complexes generated over time were quantified chromogenically. Generation of plasmin-α2-AP complexes and consumption of plasminogen were quantified by ELISA. Plasmin generation was decreased in the presence of UFH and ATH, whereas LMWH had no effect. Neither plasminogen consumption nor generation of plasmin-α2-AP complexes were affected by UFH or ATH. However, plasmin-α2-M complexes were slightly reduced by ATH suggesting that ATH may be able to compete with α2-M for plasmin. Plasmin generation may be mildly inhibited by heparin-based anticoagulants; however, heparin-catalyzed antithrombin activity is not a major inhibitor of plasmin, as compared to its natural inhibitors α2-AP and α2-M. This adds to our understanding of ATH mechanisms of action and aids in its development for clinical use.

Chemical–Physical Characterization of Polyurethane Catheters Modified with a Novel Antithrombin-Heparin Covalent Complex

Detailed structural studies were made of polyurethane catheter surfaces modified with a covalent antithrombin-heparin (ATH) complex that has superior anticoagulant activity compared to unfractionated heparin. ATH was grafted onto polyurethane catheters by surface film preparation involving a three-step process: (1) activation of ATH through functionalized poly(ethylene glycol) (PEG), (2) base-coating treatment of the polyurethane surface and (3) final attachment of ATH onto the surface by free radical polymerization. With the application of base coating, composed of polyhydroxyethylmethacrylates and poly(ethylene oxide) (PEO), the coating process could easily be transferred to other biomaterials by adjusting the base-coating composition. Anti-factor Xa assays confirmed high anticoagulant activity of the ATH coatings. To determine structural aspects critical for biological function, the product was analyzed using differential scanning calorimetry and SDS-PAGE. Radiolabeled ATH was used to determine the graft density, homogeneity and stability of modified surfaces, as well as the competition of PEO-ATH migration to the surface with self-aggregation of the PEO-ATH molecules during the coating process. X-ray photoelectron spectroscopy was used to investigate the surface chemical composition before and after ATH application. Analysis showed that PEO-ATH was strongly surface-bound at a final density of 15–200 pmol/cm2, depending on the incubation concentration.

Antithrombin-heparin covalent complex reduces microemboli during cardiopulmonary bypass in a pig model

AbstractTranscranial Doppler-detected high-intensity transient signals (HITS) during cardiopulmonary bypass (CPB) surgery have been associated with postoperative neurocognitive dysfunction, suggesting microemboli in the brain could be a contributing factor. HITS occur despite administration of unfractionated heparin (UFH). This study was done to determine whether antithrombin-heparin covalent complex (ATH), a more potent anticoagulant than heparin, can reduce HITS during CPB. In a pig CPB model, ATH, UFH, or UFH + antithrombin (AT) was intravenously administered to female Yorkshire pigs after sternotomy. Twenty minutes later, hypothermic CPB was initiated and continued for 1.25 hours, then normothermia was re-established for 45 minutes. Protamine sulfate was given to neutralize the anticoagulants, and pigs were allowed to recover. HITS were monitored using an arterial flow probe placed over the carotid artery. Compared with UFH (300 or 1000 U/kg), ATH reduced the number of HITS during CPB in a dose-dependent manner. AT (3 mg/kg) + UFH (300 U/kg) resulted in an intermediate HITS rate between UFH and ATH (2 mg/kg in terms of AT). Examination of brain sections for emboli formation confirmed that, similar to HITS, number of thrombi decreased in direct proportion to ATH dosage. These results support the hypotheses that the majority of HITS represent thromboemboli and that ATH reduces emboli formation during CPB.

Anticoagulant mechanisms of covalent antithrombin-heparin investigated by thrombelastography

We have developed an antithrombin-heparin covalent complex (ATH) which inhibits coagulation enzymes by two mechanisms: directly, or by catalytic activation of plasma antithrombin (AT). Anticoagulation by ATH was compared to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) using a blood-based, tissue factor (TF)-activated thrombelastography (TEG) assay. Simplified TEG assays with plasma or purified plasma components were used to determine the contribution of the direct and catalytic mechanisms to ATH efficacy. Low anti-Xa concentrations of UFH inhibited clot formation significantly more than equivalent concentrations of ATH or LMWH in blood and plasma. ATH had reduced ability to catalyse AT-mediated thrombin (IIa) inhibition compared to UFH. However, at high anti-Xa concentrations, ATH had similar anticoagulant activity to UFH. ATH and non-covalent AT+UFH directly inhibited clotting to a similar degree in AT-deficient plasma. IIa-ATH complexes, which are limited to catalytic inhibition, displayed impaired anticoagulation compared to free ATH, and the magnitude of this effect increased significantly as anticoagulant concentration increased. Kinetic experiments indicated that the rate of reaction of AT with IIa is lower when catalysed by ATH versus UFH. In conclusion, at low anti-Xa doses catalytic inhibition is the primary mechanism of ATH anticoagulation, and the catalytic potential of ATH is reduced relative to UFH. However, the direct inhibitory activity of ATH is comparable to non-covalent AT+UFH, and at high anti-Xa doses the direct inhibitory activity of ATH may play a larger role in anticoagulation.

In vivo rabbit acute model tests of polyurethane catheters coated with a novel antithrombin-heparin covalent complex

Catheter use has been associated with an increased risk of thrombotic complications. The objective was to make catheters less thrombogenic with the use of antithrombin-heparin covalent complex (ATH). The antithrombotic activity of ATH-coated catheters was compared to uncoated (control) and heparincoated catheters in an acute rabbit model of accelerated occluding clot formation. Anaesthetized rabbits were pre-injected with rabbit (125)I-fibrinogen, followed by insertion of test catheters into the jugular vein. Blood was drawn and held in a syringe, reinjected, then flushed with saline. The experiment was terminated when blood could no longer be withdrawn (occluding clot). Efficacy was defined as the ability of catheters to remain unoccluded. Clot formation, determined by measuring deposition of radiolabeled fibrin, was a secondary endpoint. ATH-coated catheters were resistant to clotting for the full 240-minute duration, while uncoated and heparin-coated catheters had an average clotting time of 78 and 56 minutes, respectively. The patency of ATH coating was dependant on intact heparin pentasaccharide sequences, rather than the chemistries of the basecoat, the PEO spacer arm, or the antithrombin (AT) protein. The ATH coating was stable to ethylene oxide sterilization, modest abrasion, protease attack, and the coating did not appear to leach off the catheter. Surface tension measurements showed that the ATH modified surface was more hydrophilic than uncoated control catheters or heparin-coated catheters. Thus, ATH-coated catheters are better at preventing clots than uncoated or heparin-coated catheters and show promise as an alternative to the currently available catheters in reducing thrombotic complications associated with its use.

Blood-compatible biomaterials by surface coating with a novel antithrombin–heparin covalent complex

Covalent antithrombin–heparin complex (ATH) was covalently grafted to a polycarbonate urethane (Corethane ®) endoluminal graft (a kind gift of Corvita Corporation) after being activated using 0.3% m/m NaOCl in 0.15 m phosphate pH 6.0. ATH graft density (1.98×10 −7 mol/m 2) was 6 times the maximum amount of unfractionated heparin (UFH) that could be bound to polycarbonate urethane surfaces. Surface-bound ATH could be stored in sterile 0.15 m NaCl at 4°C for at least 2 months with good antithrombotic activity before being implanted into rabbits. Analysis of ATH-coated tubing showed that it contained significant direct thrombin inhibitory activity. In vivo testing in a rabbit model was compared to non-activated non-coated surfaces, activated-non-coated surfaces, hirudin-coated surfaces and antithrombin (AT)-coated surfaces. The weight of the clot generated in the ATH-coated graft tubing was significantly less than the weight of the clot generated within the hirudin-coated graft ( p=0.03 with a 1-tailed Student's t test). The anticoagulant nature of ATH grafts in vivo was shown to be due to bound ATH because both the AT-coated surfaces and non-coated but activated surfaces showed similar thromboresistant efficacy to that of untreated material (ANOVA; p<0.05). Apart from the direct antithrombin activity that contributed to much of the prolonged patency in vivo, surface-bound ATH likely catalyzed AT inhibition of thrombin, as evidenced by a significant number of 125I-AT binding sites (⩾1.5×10 −8 mol/m 2). Thus, ATH appears to be a good candidate for coating cardiovascular devices, such as endoluminal grafts, with high levels of substitution and significant long-term blood-compatibility.

A novel antithrombin-heparin covalent complex

Heparin has been used extensively for prophylaxis and treatment of deep vein thrombosis. However, heparin has several limitations including a short intravenous half-life, inability to inhibit clot-bound thrombin, and bleeding. We have developed a covalent antithrombin-heparin complex (ATH) that has a longer intravenous half-life and greater anticoagulant activity than heparin. The antithrombotic activity of ATH was tested in a rabbit jugular vein thrombosis treatment model. Administration of ATH caused a 17% reduction in clot weight compared with an increase of 24, 60, 172 and 135% for administration of antithrombin plus heparin, heparin, antithrombin and saline, respectively. Clot weight and fibrin accretion were both significantly lower in the ATH group than in the antithrombin plus heparin group (P < 0.05). The peak anti-factor-Xa activity was fourfold higher in the ATH group than in the antithrombin plus heparin group. Using a rabbit bleeding ear model, there was no significant difference in cumulative blood loss between ATH and antithrombin plus heparin groups, at similar plasma anti-factor-Xa levels. In conclusion, ATH has superior antithrombotic activity and similar bleeding effect compared with heparin on a mass basis. The enhanced antithrombotic activity of ATH may be a result of its increased anticoagulant activity or its longer half life, or both.

Effect of a novel covalent antithrombin-heparin complex on thrombin generation on fetal distal lung epithelium.

Respiratory distress syndrome is characterized by fibrin deposition in the lung. Fibrin adversely affects surfactant function and stimulates proliferation of fibroblasts. There is evidence that these properties may be important to the development of bronchopulmonary dysplasia. Despite successful initial treatment of neonatal respiratory distress syndrome with surfactant, the incidence of bronchopulmonary dysplasia has not decreased. In previous studies, it has been demonstrated that rat fetal distal lung epithelium (FDLE) possesses both procoagulant and anticoagulant properties. In this report, we have demonstrated (using factor VII-deficient plasma) that tissue factor is expressed on the FDLE surface and promotes thrombin generation. To regulate thrombin within this procoagulant environment, we have developed a novel anticoagulant, antithrombin-heparin covalent complex (ATH) that can be retained within the lung after intrapulmonary instillation. We have demonstrated that ATH was superior to antithrombin plus standard heparin in suppressing thrombin generation (P < 0.001) and prothrombin consumption (P < 0.01) in recalcified defibrinated plasma on the surface of FDLE. Further studies with ATH in vivo need to be performed.