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To prevent coagulation in contact with blood, polydimethylsiloxane (PDMS) was modified with an antithrombin-heparin (ATH) covalent complex using polyethylene glycol (PEG) as a linker/spacer. Using NHS chemistry, ATH was attached covalently to the distal chain end of the immobilized PEG linker. Surfaces were characterized by contact angle and X-ray photoelectron spectroscopy; attachment was confirmed by decrease in contact angles and an increase in nitrogen content as determined by X-ray photoelectron spectroscopy. Protein interactions in plasma were investigated using radiolabeled proteins added to plasma as tracers, and by immunoblotting of eluted proteins. Modification of PDMS with PEG alone was effective in reducing non-specific protein adsorption; attachment of ATH at the distal end of the PEG chains did not significantly affect protein resistance. It was shown that surfaces modified with ATH bound antithrombin selectively from plasma through the pentasaccharide sequence on the heparin moiety of ATH, indicating the ability of the ATH-modified surfaces to inhibit coagulation. Using thromboelastography, the effect of ATH modification on plasma coagulation was evaluated directly. It was found that initiation of coagulation was delayed and the time to clot was prolonged on PDMS modified with ATH/PEG compared to controls. For comparison, surfaces modified in a similar way with heparin were prepared and investigated using the same methods. The data suggest that the ATH-modified surfaces have superior anticoagulant properties compared to those modified with heparin.

Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with limitations, such as the inability to inhibit surface bound coagulation factors. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties compared with UFH. Advantages of ATH include enhanced inhibition of surface-bound coagulation enzymes and the ability to reduce the overall size and mass of clots in vivo. The interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Our study utilised discontinuous second order rate constant (k₂) assays to compare the rates of inhibition of free and fibrin-associated plasmin by AT+UFH vs ATH. Additionally, we evaluated the effects of AT+UFH and ATH on plasmin generation in the presence of fibrin. The k₂ values for inhibition of plasmin were 5.74 ± 0.28 x 10⁶ M⁻¹ min⁻¹ and 6.39 ± 0.59 x 10⁶ M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. In the presence of fibrin, the k₂ values decreased to 1.45 ± 0.10 x 10⁶ M⁻¹ min⁻¹ and 3.07 ± 0.19 x 10⁶ M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. Therefore, protection of plasmin by fibrin was observed for both inhibitors; however, ATH demonstrated superior inhibition of fibrin-associated plasmin. Rates of plasmin generation were also decreased by both inhibitors, with ATH causing the greatest reduction (approx. 38-fold). Nonetheless, rates of plasmin inhibition were 2-3 orders of magnitude lower than for thrombin, and in a plasma-based clot lysis assay ATH significantly inhibited clot formation but had little impact on clot lysis. Cumulatively, these data may indicate that, relative to coagulant enzymes, the fibrinolytic system is spared from inhibition by both AT+UFH and ATH, limiting reduction in fibrinolytic potential during anticoagulant therapy.

Factor (F)Xa within the prothrombinase complex is protected from inhibition by unfractionated heparin (UFH), enoxaparin and fondaparinux. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. We have also demonstrated that ATH is superior at inhibiting coagulation factors when assembled on artificial surfaces. The objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit FXa within the prothrombinase complex when the enzyme complex is assembled on the more native platelet system. Discontinuous inhibition assays were performed to determine final k2-values for inhibition of FXa, FXa within the platelet-prothrombinase, or FXa within prothrombinase devoid of various components. Thrombin generation and plasma clotting was also assayed in the presence of resting/activated platelets ± inhibitors. Protection of FXa was not observed for ATH, whereas a moderate 60% protection was observed for AT+UFH. ATH inhibited platelet-prothrombinase ~4-fold faster than AT+UFH. Relative to intact prothrombinase, ratesfor FXa inhibition by AT+UFH in prothrombinase complexes devoid of either prothrombin (II)/activated platelets/FVa were higher. However, inhibition by AT+UFH of prothrombinase devoid of FII yielded slightly lower rates compared to free FXa inhibition. Thrombin generation and plasma clotting was enhanced with activated platelets, while inhibition was better by ATH compared to AT+UFH, thus suggesting an overall enhanced anticoagulant activity of ATH against platelet-bound prothrombinase complexes.

The role of red blood cells (RBCs) in coagulation is not well understood. Overt exposure of phosphatidylserine on surfaces of RBCs provide docking sites for formation of the prothrombinase complex, which further aids in amplification of coagulation leading to subsequent thrombosis. No studies to date have evaluated heparin inhibition of the RBC-prothrombinase system. Therefore, this study examines the ability of heparin and a covalent antithrombin-heparin complex (ATH) to inhibit the RBC-prothrombinase system. Discontinuous inhibition assays were performed to obtain k₂ values for inhibition of free or prothrombinase-bound Xa by antithrombin and unfractionated heparin (AT + UFH) versus ATH. In addition, components of the complex (prothrombin, RBCs or Va) were excluded prior to reaction with inhibitors to investigate potential mechanisms involved. Inhibition of thrombin generation, fibrinogen conversion and plasma clotting by the RBC-prothrombinase system was also examined. Protection of Xa was observed for AT + UFH and not for ATH reactions. Inhibition rates for ATH were significantly faster when compared with AT + UFH results. The greatest impact on Xa inhibition was observed from factor Va omission for both inhibitors. ATH inhibited thrombin generation, fibrinogen conversion and plasma clotting better compared with AT + UFH. This study determined potential control of coagulation contributed by RBCs. Moreover, greater control of coagulation is achieved by covalently linking heparin to AT.

A segmented polyurethane (PU) was modified with polyethylene oxides (PEO) of varying molecular weight and end group. The PEO served as linker/spacers to immobilize an antithrombin-heparin (ATH) anticoagulant complex on the PU. Isocyanate groups were introduced into the PU to enable attachment of either "conventional" homo-bifunctional dihydroxy-PEO (PEO-OH surface) or a hetero-bifunctional amino-carboxy-PEO (PEO-COOH surface). The PEO surfaces were functionalized with N-hydroxysuccinimide (NHS) groups using appropriate chemistries, and ATH was attached to the distal NHS end of the PEO (PEO-OH-ATH and PEO-COOH-ATH surfaces). Water contact angle and fibrinogen adsorption measurements showed increased hydrophilicity and reduced fibrinogen adsorption from buffer on all PEO surfaces compared to unmodified PU. ATH uptake on NHS-functionalized PEO was quantified by radiolabeling. Despite the different PEO molecular weights and end groups, and NHS-functionalization chemistries, the surface densities of ATH were similar. The adsorption of fibrinogen and antithrombin (AT) from plasma was measured in a single experiment using dual radiolabeling. On PEO-ATH surfaces fibrinogen adsorption was minimal while AT adsorption was high showing the selectivity of the heparin moiety of ATH for AT. The PEO-COOH-ATH surfaces showed slightly greater AT adsorption than the PEO-OH-ATH surfaces. Thrombin adsorption on all of the PEO-ATH surfaces was greater than on the corresponding PEO surfaces without ATH, and was highest on the PEO-OH-ATH, suggesting potential anticoagulant properties for this surface via direct thrombin inhibition by the AT portion of ATH., (Copyright © 2012 Wiley Periodicals, Inc.)

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.

Polyurethane (PU) was modified using isocyanate chemistry to graft polyethylene oxide (PEO) of various molecular weights (range 300-4600). An antithrombin-heparin (ATH) covalent complex was subsequently attached to the free PEO chain ends, which had been functionalized with N-hydroxysuccinimide (NHS) groups. Surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS) to confirm the modifications. Adsorption of fibrinogen from buffer was found to decrease by ~80% for the PEO-modified surfaces compared to the unmodified PU. The surfaces with ATH attached to the distal chain end of the grafted PEO were equally protein resistant, and when the data were normalized to the ATH surface density, PEO in the lower MW range showed greater protein resistance. Western blots of proteins eluted from the surfaces after plasma contact confirmed these trends. The uptake of ATH on the PEO-modified surfaces was greatest for the PEO of lower MW (300 and 600), and antithrombin binding from plasma (an indicator of heparin anticoagulant activity) was highest for these same surfaces. The PEO-ATH- and PEO-modified surfaces also showed low platelet adhesion from flowing whole blood. It is concluded that for the PEO-ATH surfaces, PEO in the low MW range, specifically MW 600, may be optimal for achieving an appropriate balance between resistance to nonspecific protein adsorption and the ability to take up ATH and bind antithrombin in subsequent blood contact., (© 2011 American Chemical Society)

The anticoagulant properties and platelet interactions of gold surfaces modified with an antithrombin-heparin (ATH) complex are reported. ATH was attached to gold through either a short disulfide (linker) or a thiol-terminated polyethylene oxide (PEO) (linker, spacer). Analogous surfaces were prepared with uncomplexed heparin. Antithrombin (AT) uptake was measured before and after selectively destroying the active pentasaccharide sequence of the heparin moiety, and was found to be predominantly through the active sequence on all of the surfaces. AT binding was higher on the ATH surfaces than on the corresponding heparin surfaces. Heparin activity was assessed by an anti-factor Xa assay. The ratio of active heparin density (from the anti-FXa assay) to total heparin density was taken as a measure of heparin bioactivity. The ratio was greater on the ATH- than on the heparin-modified surfaces, i.e. the PEO-ATH surfaces showed the greater proportion of active heparin. Platelet adhesion from flowing whole blood was found to be reduced on PEO- and ATH-modified surfaces compared to bare gold. The PEO-ATH modified surfaces, but not the heparinized surfaces, were shown to prolong the clotting time of recalcified plasma., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Transcranial 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.

Protein C in its activated form (APC) limits thrombin generation. Protein C inhibitor (PCI) readily neutralizes APC. Heparin accelerates this reaction, which may complicate anticoagulant treatment in patients with varying APC generation potential. A potent anticoagulant conjugate of antithrombin and heparin (ATH) was prepared, and its effect on APC+PCI reactions was tested. Second order rate constants for APC+PCI reactions were measured by discontinuous rate experiments in the presence of heparin or ATH. Similarly, low molecular weight fractions of heparin (LMWH) and ATH (LMWATH) were tested, as was high molecular weight ATH (HMWATH). Mechanisms of heparin or ATH binding to APC or PCI were assessed using electrophoresis. While heparin gave a higher maximal APC inhibition rate compared to ATH, peak inhibition rate was achieved at comparatively lower ATH concentrations. Since LMWH was ineffective at enhancing APC inhibition by PCI, unfractionated heparin likely acts by bridging APC and PCI. Unlike heparin, ATH may conformationally activate either APC or PCI since LMWATH significantly catalyses APC inhibition. Binding studies showed that ATH readily associates with APC. Thus, although a small fraction of ATH efficiently catalyses APC inhibition by PCI, complete ATH preparations induce a decreased maximal rate of APC-PCI formation compared to unfractionated heparin.

Gold was used as a substrate for immobilization of an antithrombin-heparin (ATH) covalent complex to investigate ATH as a surface modifier to prevent blood coagulation. Three different surface modification methods were used to attach ATH to gold: (i) direct chemisorption; (ii) using dithiobis(succinimidyl propionate) (DSP) as a linker molecule and (iii) using polyethylene oxide (PEO) as a linker/spacer. The ATH-modified surfaces were compared to analogous heparinized surfaces. Water contact angles and X-ray photoelectron spectroscopy confirmed the modifications and provided data on surface properties and possible orientation. Ellipsometry measurements showed that surface coverage of DSP and PEO was high. ATH and heparin densities were quantified using radioiodination and quartz crystal microbalance, respectively. The surface density of ATH was greatest on the DSP surface (0.17 microg cm(-2)) and lowest on the PEO (0.05 microg cm(-2)). The low uptake on the PEO surface was likely due to the protein resistance of the PEO component. Using radioiodinated antithrombin (AT), it was shown that ATH-immobilized surfaces bound significantly greater amounts from both buffer and plasma than the analogous heparinized surfaces. Immunoblot analysis of proteins adsorbed from plasma demonstrated that surfaces chemisorbed with PEO, whether or not subsequently modified with ATH, inhibited non-specific adsorption. The immunoblot response for AT was stronger on the DSP-ATH than on the heparin surfaces, thus confirming the results from radiolabelling. The ATH surfaces again showed higher selectivity for AT binding than analogous heparin-modified surfaces, indicating the enhanced anticoagulant potential of ATH for biomaterial surface modification., (Copyright 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Thrombomodulin (TM), which variably contains a chondroitin sulfate (+/-CS), forms an anticoagulant complex with thrombin (IIa). IIa-TM(+/-CS) converts protein C (PC) into activated PC (APC), which then inactivates activated factors V (FVa) and VIII (FVIIIa). This reduces prothrombinase and tenase complexes that generate IIa. Heparin (H) increases the rate of IIa-TM inhibition by antithrombin (AT) and enhances FV cleavage by APC. Our novel covalent AT-H (ATH) product, has superior anticoagulant activity compared to AT + unfractionated H (UFH). We studied mechanisms by which ATH versus AT + UFH inhibits IIa-TM(+/-CS), and ATH influences on APC cleavage of FV/FVa compared to UFH. Findings would determine how these reactions moderate ATH's overall effects as an anticoagulant. Discontinuous second order rate inhibition assays of IIa-TM(+/-CS) inhibition by AT + UFH or ATH were performed in presence or absence of human umbilical vein endothelial cells (HUVECs). FV/FVa cleavage by APC in the presence of UFH or ATH was analysed by Western blots. ATH increased IIa-TM(+/-CS) inhibition to a greater degree than AT + UFH, both on plastic and HUVEC surfaces. Unlike UFH, ATH did not accelerate FV cleavage by APC, but ATH did enhance FVa cleavage relative to UFH. Increased IIa-TM inhibition by ATH downregulates PC activation. However, ATH does accelerate downstream inactivation of FVa, which increases its potency for IIa generation inhibition compared to UFH. This trend holds true in the presence of APC's cofactor, protein S. Overall, ATH may have a balanced function towards inhibiting or accelerating PC pathway activities.

Antithrombin (AT) is a native plasma protein that acts as the main inhibitor of enzymes generated by the coagulation cascade. In extreme thrombotic conditions, consumption of plasma AT can make treatment with AT-associated heparin therapies less effective. Supplementation with recombinant human AT (rhAT) has shown promise but altered pharmacokinetics were observed when comparing stable heparin complexes of the plasma-derived AT (pAT) and rhAT proteins. To understand the differential clearance mechanisms, biodistribution of rhAT and pAT was determined. (125)I-labelled ATryn (rhAT) or Kybernin P (pAT) was intravenously injected into rabbits. At various time points, animals were sacrificed and organs analysed for bound radioactivity. (131)I-albumin, injected shortly before termination, was used as a marker for trapped blood. Levels of circulating protein + metabolites were significantly less for rhAT than pAT (p < 0.001) and removal of acid soluble fragments confirmed that differences were due to more rapid rhAT clearance. More rhAT (28% dose) than pAT (3% dose) was liver-associated by the earliest time points, corresponding to elevated rhAT degradation products in urine/feces. However, at intermediate times (4 hours), rhAT showed significantly increased arterial and venous uptake over pAT (p

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.

Since most thrombotic reactions occur on the vessel wall, interaction of anticoagulants with vascular components is critical. Heparin (H) is the primary drug for treatment and prevention of thrombosis. To improve H's efficacy and bioavailability, a covalent complex of H and its biological target, antithrombin (AT), was developed. While H has a short, variable intravenous half-life leading to unpredictable anticoagulation, clearance of covalent ATH complex is slower. H's variable anticoagulant effect arises from interactions with plasma and vessel wall proteins. ATH has increased bioavailability due to lower plasma protein and endothelial binding relative to H. Pharmacodynamic studies demonstrate that the AT moiety can regulate ATH binding to target tissues. For example, blood vessel binding is enhanced using ATH containing recombinant AT with oligomannose structures that can interact with endothelial mannose receptor lectins. Furthermore, recent work has shown that inhibition of factor VIIa/tissue factor complex by ATH is significantly faster than AT + H. Similarly, thrombin bound to endothelial thrombomodulin is inhibited more efficiently by ATH than by AT + H, which might improve regulation of thrombin generation. Overall, linking H to AT may prevent unwanted protein interactions and allow vessel wall sites to be targeted. This review examines ATH biodistribution.

We have developed a potent antithrombin (AT)-heparin conjugate (ATH) that is retained in the lung to prevent pulmonary thrombosis associated with respiratory distress in premature newborns. During continuing maturation, pulmonary angiogenesis in premature infants would be a crucial process in lung development. A naturally occurring latent form of antithrombin (L-AT) has antiangiogenic effects on lung vascularization. However, impact of latent ATH (L-ATH) on developing lung vascularization is unknown. Thus, effects of L-AT and L-ATH on fetal murine lung development were compared. Lung buds from embryonic day 11.5 (E11.5) Tie2-LacZ mouse embryos were incubated in DMEM plus FBS supplemented with PBS, AT, L-AT, heparin, ATH, or L-ATH. Vasculature of cultured explants was quantified by X-galactosidase staining. RNA was analyzed with murine gene probes for angiopoietin (Ang)-1, Ang-2, fibroblast growth factor 2 (FGF2), platelet endothelial cell adhesion molecule (PECAM), and vascular endothelial growth factor (VEGF). FGF2-supplemented medium was used to test contribution to effects of L-AT and L-ATH on angiogenesis. Epithelial branching morphogenesis was inhibited by L-AT (P = 0.003) and heparin (P < 0.001). L-AT and heparin decreased relative vascular area compared with PBS, ATH, and L-ATH. Expressions of all genes studied were downregulated by L-AT. However, L-AT and L-ATH inhibited branching morphogenesis and vasculature with added FGF2. These findings indicate that covalent linkage of AT to heparin negates disruptive effects of these moieties on lung morphology, vascularization, and growth factor gene expression. ATH may have enhanced safety as an anticoagulant during vascular development.

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) have been utilized as primary anticoagulants for thrombosis prophylaxis and treatment. However, a number of biophysical and safety limitations have led to development of new anticoagulants. Covalent antithrombin-heparin (ATH) complexes may address many of these issues. Early ATH products were prepared that had increased intravenous half-lives relative to UFH but lacked any improvement in anti-factor Xa activity or had no catalytic activity or reactivity against thrombin. However, a recent conjugate developed by Chan et al. has displayed a number of superior properties. Chan et al. ATH has an increased direct thrombin inhibition rate and can catalyze coagulant enzyme inhibition by exogenous antithrombin with very high specific activity. Unlike UFH, clot-bound thrombin is readily inhibited by ATH and, at similar antithrombotic efficacy, the ATH has improved bleeding profiles compared to heparins. Given the preclinical findings, Chan et al. ATH may warrant clinical trial testing for control of clot propagation.

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.

We have developed a covalent antithrombin-heparin (ATH) complex with advantages compared to non-covalent antithrombin:heparin (AT:H) mixtures. In addition to increased activity, ATH has a longer intravenous half-life that is partly due to reduced plasma protein binding. Given ATH's altered clearance, we investigated biodistribution of ATH in vivo. ATH made from either human plasma-derived AT (pATH) or recombinant human (produced in goats) AT (rhATH) was studied. 125I-ATH + unlabeled carrier was injected into rabbits at different doses. 131I-labeled albumin was administered just before sacrifice as a marker for trapped blood in tissues. Immediately after sacrifice, animal components were removed, weighed, and subsamples were counted for gamma-radioactivity. Percent recoveries of ATH in various organs/compartments at different time points were calculated, and kinetic distribution plots generated. At saturating doses, early disappearance of rhATH from the circulation was much faster than pATH. Co-incident with clearance, 26 +/- 3% of dose for rhATH was liver-associated compared to only 3.7 +/- 0.5% for pATH by 20 min. Also, at early time periods, >60% of all extravascular ATH was liver-associated. Analysis of the vena cava and aorta suggested that vessel wall binding might also account for initial plasma loss of rhATH. By 24 h, most of pATH and rhATH were present as urinary degradation products (51 +/- 3% and 63 +/- 8%, respectively). In summary, systemic elimination of ATH is greatly influenced by the form of AT in the complex, with liver uptake and degradation playing a major role.

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.

Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.

Numerous studies have shown that fibrin-bound thrombin (IIa) is protected from inhibition by antithrombin (AT) + heparin (H) due to the formation of a ternary fibrin.IIa.H complex. We investigated factors affecting the inhibition of fibrin.IIa by a covalent complex of AT and H (ATH). The rate of IIa reaction with ATH was decreased 2-3-fold by fibrin monomer as compared to 57-fold for AT + heparin with high AT affinity. Furthermore, although the reaction of AT + H with a IIa mutant with decreased H binding (RA-IIa) was inhibited 2-3-fold in the presence of fibrin, reaction rates of ATH + RA-IIa were not reduced by fibrin. The relative difference in the effect of fibrin on the ATH reaction with RA-IIa compared to that for reactions of AT + H with RA-IIa is consistent with the fact that, in the absence of fibrin, the rate of the ATH reaction with RA-IIa relative to IIa was much less reduced (8-fold) compared to the corresponding reactions of AT + H (decreased 306 fold). Similarly, the addition of excess H in the absence of fibrin gave only a small decrease in rate of ATH + IIa reaction. However, in the presence of fibrin, the addition of 40-fold excess H decreased the rate of ATH inhibition of IIa by 1 order of magnitude. Experiments with ATH containing low molecular weight heparin chains with low AT affinity showed that IIa inhibition requires ATH with long chains that activate the AT moiety. Finally, electrophoresis of fibrin +/- ((125)I-)IIa +/- ((125)I-)ATH on native and denaturing gels showed that ATH forms ATH-IIa complexes that remain bound to fibrin through the ATH component. Thus, ATH is a potent inhibitor of fibrin-bound IIa, likely due to the formation of fibrin.ATH-IIa as opposed to fibrin.IIa.H ternary complexes.

Background: The anticoagulant effect of heparinoids is attributed to their cofactor activity for antithrombin (AT) and heparin cofactor II. In patients with thrombosis, however, thrombin is often protected from AT-dependent, heparin-mediated inactivation. The purpose of this study was to compare the properties of unfractionated/standard heparin (UFH/SH) and those of a novel covalent AT-heparin complex (ATH) in a rabbit arterial thrombosis prevention and bleeding model., Methods and Results: Thrombosis in the distal aorta was triggered by vessel wall injury and critical stenosis. Blood flow in the damaged arterial segment was monitored with a flow probe placed distal to the constrictor. Rabbits were given doses of SH (62.5 to 187.5 IU x kg(-1) x 90 min(-1)) or ATH (16 to 65 IU x kg(-1) x 90 min(-1)). Cumulative blood loss from skin incisions was used to assess drug safety. The antithrombotic effects of ATH were greater than those of SH as measured by clot weight, blood flow, and vessel patency; eg, complete thrombus resolution was achieved with ATH (33 to 65 IU/kg), but not SH (125.0 to 187.5 IU/kg). At doses that produced equivalent vessel patency (50% to 60%), blood loss induced by ATH (60.2 microL) was 2.6-fold lower (P<0.05) than that induced by SH (154.6 microL)., Conclusions: In our experimental system, ATH was able to control arterial thrombosis more effectively than its SH precursor, without pronounced bleeding.

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 x 10(-7) mol/m2) 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 degrees 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 boththe 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 (> or = 1.5 x 10(-8) mol/m2). 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.

Recently, we developed a covalent antithrombin-heparin complex (ATH) as a possible treatment for respiratory distress syndrome. ATH reacted rapidly with thrombin and efficiently catalyzed the inhibition of either thrombin or factor Xa by exogenous antithrombin. In order to investigate mechanisms for the conjugate's unusual anticoagulant properties, changes in fluorescence due to covalent linkage or addition of exogenous antithrombin were studied in relation to reaction with thrombin derivatives or factor Xa. The emission spectrum of ATH was similar to that of antithrombin plus heparin mixtures. ATH quickly inhibited thrombin or factor Xa activities, as measured by a fluorogenic substrate. Fluorescein-labeled heparin was displaced from either thrombin or active site blocked thrombin by ATH, indicating that thrombin must bind to the conjugate's heparin moiety. Interaction of thrombin with ATH's heparin component was confirmed by a slow reaction rate of conjugate with a thrombin mutant that has weak heparin binding. Total intrinsic fluorescence increased when exogenous antithrombin was added to ATH, indicating that the catalytic mechanism may occur through a second inhibitor binding site. Thus, ATH reacts directly with thrombin through a bridge mechanism and probably catalyzes the reaction of thrombin with antithrombin by a second binding sequence on its heparin chain.

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.