Covalent Antithrombin-heparin 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.

Surface modification of poly(dimethylsiloxane) with a covalent antithrombin-heparin complex for the prevention of thrombosis: use of polydopamine as bonding agent.

A modified poly(dimethyl siloxane) (PDMS) material is under development for use in an extracorporeal microfluidic blood oxygenator designed as an artificial placenta to treat newborn infants suffering from severe respiratory insufficiency. To prevent thrombosis triggered by blood-material contact, an antithrombin-heparin (ATH) covalent complex was coated on PDMS surface using polydopamine (PDA) as a "bioglue". Experiments using radiolabelled ATH showed that the ATH coating on PDA-modified PDMS remained substantially intact after incubation in plasma, 2% SDS solution, or whole blood over a three day period. The anticoagulant activity of the ATH-modified surfaces was also demonstrated: in contact with plasma the ATH-coated PDMS was shown to bind antithrombin (AT) selectively from plasma and to inhibit clotting factor Xa. It is concluded that modification of PDMS with polydopamine and ATH shows promise as a means of improving the blood compatibility of PDMS and hence of the oxygenator device.

PO-0759 Surface Modification Of A Polydimethylsiloxane Microfluidic Oxygenator With Dopamine And A Covalent Antithrombin-heparin Complex For The Prevention Of Thrombosis

<h3>Introduction</h3> Prematurely born infants suffer respiratory insufficiency, our lab has developed a novel microfluidic oxygenator with polydimethylsiloxane (PDMS) gas transfer membranes to provide respiratory support. The objective of the work reported here was to modify the PDMS surfaces with a covalent antithrombin-heparin (ATH) complex to prevent coagulation and thrombosis due to blood-material contact. Specifically the ATH was attached to the PDMS using dopamine as a ‘bio-glue’. <h3>Materials and methods</h3> PDMS discs were incubated in a solution of dopamine hydrochloride and then in ATH solution. A separate set of PDMS discs were justed incubated in ATH. Uptake of ATH and adsorption of antithrombin (AT) from plasma (a measure of anticoagulant activity) to the various surfaces was measured using ¹²⁵I-labelled ATH and AT. Stability of ATH on surfaces was evaluated by measuring residual radioactivity after incubation in blood. <h3>Results</h3> ATH uptake on PDMS was higher with dopamine as glue (Fig. 1), ~74% of the original ATH was lost from PDMS+ATH after 3 h in blood, whereas only ~30% was lost from PDMS+DOP+ATH. The ATH surface with dopamine is adhesive, thus showed higher AT adsorption (42.3 ng/cm²) compared to PDMS (6.3 ng/cm²), and therefore should have higher anticoagulant activity. <h3>Conclusions</h3> An antithrombin-heparin complex (ATH) was attached to PDMS using dopamine as a bio-glue. The use of dopamine gave surfaces with higher concentration and greater stability of ATH. The bound ATH showed potential for anticoagulant activity through extensive adsorption of antithrombin from plasma.

Surface modification of polydimethylsiloxane with a covalent antithrombin–heparin complex to prevent thrombosis

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.

Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets

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.

Interactions of Heparin and a Covalently Linked Antithrombin Heparin Complex with the Components of the Fibrinolytic Pathway.

Abstract 2211 Introduction:Unfractionated heparin (UFH) is used for the prophylaxis and treatment of thromboembolic diseases. UFH catalyzes inhibition by antithrombin (AT) of the serine proteases in the coagulation cascade. Additionally, UFH has been shown to interact with components of the fibrinolytic pathway in vitro. However UFH has several limitations which impact its utility as a therapeutic agent. Our lab has developed a novel covalent antithrombin-heparin complex (ATH) which inhibits most serine proteases of the coagulation pathway significantly faster when compared to non covalent mixtures of AT and UFH. However, the interactions of ATH with the components of the fibrinolytic pathway have not been studied before. Thus, the present study investigates possible serpin-heparin interactions of AT + UFH vs ATH within the fibrinolytic pathway. Methods:Discontinuous second order rate constant assays under pseudo-first order conditions were carried out to obtain second order rate constant (k2) values for the inhibition of plasmin by AT+UFH versus ATH. Briefly, at specific time intervals 20 nM plasmin was inhibited by 200 nM AT + 0–5000 nM UFH or by 200 nM ATH in the presence of 2.5 mM Ca2+. Reactions were neutralized by the simultaneous addition of a solution containing polybrene and plasmin substrate S-2366™ in buffer. Residual plasmin activity was measured and the final k2 values calculated. For experiments involving tPA, wells containing 40nM tPA and increasing concentrations of AT, UFH or ATH, at mole ratios ranging from 0 to 20:1, were incubated for 15 min. Reactions with tPA were neutralized by simultaneous addition of a solution containing either polybrene and tPA substrate, S-2288™ in buffer, (ATH and UFH) or only the substrate S-2288™ in buffer (AT). Enzyme activity was then determined by measuring rate of substrate cleavage (Vmax). Results:When plasmin was inhibited by AT in the absence of UFH, k2 values of 2.82×105 +/− 4.46×104 M−1 min−1 were observed. The k2 values increased with addition of successively higher concentrations of UFH up to a plateau with maximal k2 of 5.74×106 +/− 2.78×105 M−1 min−1 at a UFH concentration of 3000nM. For inhibition of plasmin by ATH, k2 values of 6.39 × 106 +/− 5.88 × 105 M−1 min−1 were observed. Inhibition of plasmin by ATH was not significantly different when compared to the highest k2 values obtained with UFH. (p=0.36) No statistically significant difference in tPA enzyme activity was observed when Vmax values for tPA alone were compared with those in the presence of AT, UFH or ATH. (p=0.932, p=0.085, p=0.31 respectively) Significance:The characteristic shape of the curve obtained from the k2vs. UFH plot suggests that the mechanism responsible for inhibition of plasmin by AT+UFH involves conformational activation of the serpin. The k2 values in this study for inhibition of plasmin by both AT+UFH and ATH were three orders of magnitude lower than k2 values for inhibition of thrombin or factor Xa. Furthermore these results suggest that tPA is not inhibited by AT + UFH or ATH, and is not influenced by the presence of UFH alone. Cumulatively, this indicates that the fibrinolytic pathway is minimally impacted by AT + UFH or ATH, allowing maximal antithrombotic potential to be achieved during anticoagulation. Overall, the favourable anticoagulant properties of ATH combined with the findings of this study strengthens the utility of the covalent conjugate over conventional UFH for the treatment of thromboembolic disorders. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Platelet-Prothrombianse by a Covalent Antithrombin-Heparin Complex.

Abstract 2212 Introduction:Factor Xa is protected within the prothrombinase complex from inhibition by heparin. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. Previously, we have shown that ATH is able to inhibit coagulation enzymes much more efficiently than regular antithrombin+heparin (AT+UFH). For example, ATH inhibited TF/VIIa ∼30-fold faster compared to AT+UFH. Furthermore, we have also demonstrated that ATH is capable of inhibiting Xa within a prothrombinase complex assembled on synthetic phospholipid vesicles better than AT+UFH. However, ATH’s effect on prothrombinase when the complex is formed on a more native system such as platelets has never been explored. Thus, the objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit Xa within the prothrombinase complex when the enzyme complex is assembled on the platelet system. Methods:Discontinuous second order rate constant assays were performed to obtain k2-values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly prepared resting platelets were subjected to inhibition analysis by first incubating them with Xa, Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells to initiate thrombin generation, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2-values calculated. For experiments requiring activated platelets, freshly isolated platelets were activated with 5 μM calcium ionophore A23187 + 4 mM CaCl2 for 15 min at room temperature. The activated platelets were then tested in inhibition assays as described above. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of AT+UFH and ATH, additional experiments were performed where components of the complex (prothrombin, activated platelets or Va) were omitted prior to reaction with inhibitors. Thrombin generation was used to assess functionality of the activated platelet-prothrombinase system in the presence of inhibitors using a thrombin-specific substrate S-2238™. Results:The k2-values (×108M−1min−1) for inhibition of free Xa or resting platelet-prothrombinase were similar for both inhibitors, although the overall inhibition rates achieved by the ATH were 2-fold faster than AT+UFH (p<0.001). Since activated platelets are required for enhanced prothrombinase function, we then compared inhibition of free vs activated platelet-prothrombinase by the two inhibitors (platelet activation was confirmed with flow cytometry using an anti CD-41 antibody). No differences were observed in the k2-values between free Xa (3.96±0.23) and activated platelet prothrombinase (3.83±0.39) for ATH reactions. However, the k2-values for inhibition of free Xa by AT+UFH was 2.37±0.32, and assembly of Xa within the activated platelet-prothrombinase resulted in a reduction in the k2-values to 0.99±0.22 (p<0.001), thus confirming a moderate 60% protection of Xa by the prothrombinase components. However, omitting the components (prothrombin, activated platelets or Va) from the complex resulted in higher k2-values (1.76±0.37, 2.29±0.26 and 2.52±0.32, respectively p<0.01) for AT+UFH, and as expected, no net effect was observed for ATH. Thrombin generation was inhibited significantly by both AT+UFH and ATH compared to the control (p<0.001), but further analysis of thrombin potential yielded greater inhibition by ATH compared to AT+UFH (p<0.05). Conclusion:In this study, we report inhibition of the prothrombinase complex on the surface of resting and activated platelets. Consistent with previous investigations, a moderate protection of Xa was observed when the activated platelet-prothrombinase was inhibited by AT+UFH. ATH on the other hand, targets and inhibits prothrombinase complexed-Xa as fast as free Xa, and at inhibition rates that were significantly faster than AT+UFH. Thus, overall the covalent conjugate enhances anticoagulation of surface-bound enzymes and offers advantages over conventional heparin for the treatment of cell-based coagulation in vivo. Disclosures:No relevant conflicts of interest to declare.

Inhibition of the prothrombinase complex on red blood cells by heparin and covalent antithrombin-heparin complex

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.

Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex

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.

Inhibition of Red Blood Cell-Prothrombinase by Heparin and a Covalent Antithrombin-Heparin Complex

Abstract 1179 Introduction:Various hematological conditions, including sickle cell anemia, beta-thalassemia and preeclampsia, have been linked with the presence of procoagulant red blood cells (RBCs). Overt exposure of phosphatidylserine (PS) on surfaces of RBCs has been a suspect for increased thrombotic complications in these patients. Asymmetrical distribution of PS at the membrane surface may provide docking sites for the formation of the procoagulant prothrombinase complex, which further aids in amplification of the coagulation cascade leading to subsequent thrombosis. In acute thrombotic complications, heparin is used as the first line of treatment in these patients. However, little is known regarding heparin's efficiency to pacify the procoagulant RBC-prothrombinase system. Therefore, we sought to determine the rate of RBC-prothrombinase inhibition by antithrombin and unfractionated heparin (AT+UFH) versus a novel covalent complex of AT and heparin (ATH). Methods:Thrombin generation was used to assess functionality of the phosphatidic acid (PA)-induced RBC-prothrombinase system, using a thrombin-specific substrate S-2238™. Discontinuous second order rate constant assays were carried out to obtain k2 values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly isolated RBCs were activated using PA and calcium for 15 min at room temperature. Activated RBCs were incubated with factors Xa and Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells and allowed to react for 3 min at 37 °C, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2 values calculated. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of heparin and ATH, additional experiments were performed where components of the complex (prothrombin, activated RBCs or Va) were removed prior to reaction with the inhibitors. Results:PA-induced RBC-prothrombinase yielded a 4-fold greater thrombin generation compared to non-activated RBCs (p<0.05). The k2 values (x 108 M−1min−1) for inhibition of Xa alone by AT+UFH was 3.14 ± 0.11, whereas inhibition of RBC-prothrombinase significantly decreased this value to 2.12 ± 0.22, p<0.02. In comparison, ATH inhibited both free Xa and prothrombinase-complexed Xa at significantly faster rates compared to AT+UFH reactions (5.12 ± 0.27 and 4.85 ± 0.24 (p<0.0001), respectively). No significant differences were observed for free Xa or RBC-prothrombinase inhibition by ATH (p<0.46). When components of the RBC-prothrombinase were removed from reactions, the greatest impact on Xa inhibition was observed from factor Va omission. A significant decrease in inhibition rate was shown for both AT+UFH and ATH (4-fold and 2-fold, respectively) due to factor Va absence. Conclusion:In this study, we report inhibition of the prothrombinase complex on the surface of procoagulant RBCs. Consistent with vesicle and activated platelet systems, RBC-prothrombinase seems to protect Xa from inhibition by AT+UFH, which suggests that heparin efficiency is also compromised when prothrombinase forms on the surface of procoagulant RBCs. Relative to AT+UFH reactions, RBC-prothrombinase was significantly enhanced and no protection of Xa was observed with ATH. From our findings, factor Va may be the major component moderating the protective effect. Overall, these data indicate a clinical advantage for ATH over heparin in treatment of blood-based coagulation in vivo. This study also provides some understanding of how heparin or ATH treatment may interact with procoagulant complexes on the surface of RBCs in patients with red cell disorders that have thrombotic complications. Disclosures:No relevant conflicts of interest to declare.

Exploring the Impact of Clinical Anticoagulants on Venous Thrombosis Stability Using a Novel Intravital Murine Model

Abstract 1246 Background:Thrombus stability influences the progression of deep vein thrombosis to a potentially fatal pulmonary embolism (PE) event. Anticoagulants are clinically administered to treat venous thrombosis. However, the effect of anticoagulants on thrombus stability remains unknown. Objective:We developed a novel intravital mouse model to explore the hypothesis that administration of clinical anticoagulants would decrease early thrombus stability, thereby potentially increasing PE risk. Methods:The trachea and jugular vein were cannulated, and the femoral vein isolated, in wild type C57/Bl6 female mice. Platelets were labeled in vivo using anti-mouse CD41 Fab fragments conjugated to Alexa Fluor-488. A 1 × 2 mm filter paper strip, saturated in 4% ferric chloride, was applied to the femoral vein for 5 minutes to induce thrombus formation. Wide-field fluorescent microscopy was used to quantify thrombus stability. Stability was related to the number of embolic events and loss of platelet intensity captured downstream of the thrombus at 5, 15, 30, 45, and 60 minutes post thrombus formation. Results:The mean number of embolic events and loss of platelet intensity decreased over time in wild type mice (n = 12). This suggested that thrombus stability increases over time. Anticoagulants were administered via a jugular vein catheter, at 12 minutes post thrombus formation, to assess impact on embolization. The anticoagulants examined were hirudin (8U/g mouse body weight), unfractionated heparin (UFH) (0.1U/g), a covalent antithrombin-heparin complex (ATH) (0.08U/g), and fondaparinux (0.1μg/g). We observed an overall a) increase in the number of embolic events and b) increase in platelet intensity lost over time in mice injected with hirudin (n = 12) and UFH (n = 12) when compared to untreated wild type control mice. The total number of embolic events occurring over one hour substantially increased in the hirudin-treated group (p = 0.09), which was also associated with an overall increase in total platelet intensity (p = 0.08), compared to untreated control mice. In addition, there was an increase in the total number of embolic events compared to the UFH-treated group (p = 0.09). Administration of hirudin, a direct thrombin inhibitor (DTI), and UFH, an indirect thrombin inhibitor, could result in decreased venous thrombus stability. However, it appears that the DTI is associated with greater thrombus instability. In the ATH-treated group (n = 12), an increase in embolic events at 15 minutes was observed, followed by a decrease in embolization. ATH could initially disrupt thrombus stability through inhibition of fibrin-bound thrombin, before acting in a stabilizing manner. Administration of fondaparinux (n = 6), an indirect factor-Xa inhibitor, demonstrated an overall decrease in embolic events and platelet intensity lost over time. When compared to control groups, there was a significant decrease in total number of embolic events and total amount of platelet intensity lost in the fondaparinux group (p < 0.05). Use of a factor-Xa inhibitor appears to enhance thrombus stability more effectively in comparison to direct and indirect thrombin inhibitors. Conclusion:Use of anticoagulants that inhibit thrombin predominantly could decrease early thrombus stability and potentially increase the likelihood of a PE event. Disclosures:No relevant conflicts of interest to declare.

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.

Investigation Into Mechanisms of Prothrombinase Complex Inhibition by a Covalent Antithrombin-Heparin Complex.

Abstract 2136Poster Board II-113 Introduction:Formation of the prothrombinase complex results in efficient generation of thrombin (IIa) by a vast enhancement of the factor Xa (Xa) reaction with prothrombin (II). However, the integration of Xa into the complex significantly decreases its availability for inhibition by antithrombin+heparin (AT+H). Our laboratory has developed a novel anticoagulant by covalently linking antithrombin to heparin (ATH). We have shown that ATH, compared to AT+H, significantly increased the rate of inhibition of clot-bound thrombin as well as several other coagulation factors in isolation. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase system. Methods:Pseudo first-order kinetic experiments for the inhibition of Xa in the prothrombinase complex were performed by reacting AT+H or ATH with CaCl2, phospholipid vesicles (PC/PS), factor Va (Va), II, specific IIa inhibitor Pefabloc and Xa in buffer. At specific time intervals, the reactions were neutralized with Na2EDTA, polybrene and Xa substrate (S-2222) in buffer, followed by measurement of residual enzyme activity. Second-order rate constants (k2) were calculated from semi-logarithmic plots of residual Xa activity remaining versus time. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of ATH, additional experiments were performed where components of the complex (II, PC/PS or Va) were removed prior to reaction with AT+H or ATH. Results:The k2value (× 108 M−1min−1) for the inhibition of Xa alone by AT+H was 1.47 ± 0.08. Incorporation of Xa into the prothrombinase complex significantly decreased this value to 0.67 ± 0.05, p<0.001. In comparison, ATH inhibited free-Xa and prothrombinase-complexed Xa at significantly faster rates than AT+H (2.83 ± 0.15 and 2.11 ± 0.38 (p<0.01), respectively). Relative to intact prothrombinase, rates for inhibition by AT+H of Xa in complexes devoid of PC/PS, Va or II significantly increased to 1.49 ± 0.08, 1.44 ± 0.08 and 1.24 ± 0.04, p<0.001, respectively. In contrast, removal of II from prothrombinase significantly decreased the k2 for Xa inhibition by ATH to 1.57 ± 0.04, p<0.05. Although removal of PC/PS from the system resulted in a higher k2 (2.79 ± 0.14, p<0.01), removal of Va resulted in no significant change (2.09 ± 0.09, p=0.79) for ATH reactions. Conclusion:Based on these results, we conclude that the prothrombinase complex hinders the inhibitory action of AT+H on Xa, whereas ATH is less affected. In fact, ATH inhibition of the prothrombinase complex was significantly higher than the inhibition rate for either free or prothrominase-bound Xa by AT+H. We speculate that, similar to inhibition of fibrin-bound thrombin, H in the non-covalent AT+H dissociate and bind to components within the prothrombinase that repel incoming AT+H and preventing Xa inhibition. These complexes cannot occur when AT is covalently linked to H, thus allowing for enhanced inhibition of prothrominase-bound Xa by ATH. Absence of II from the prothrombinase complex removes interference of AT+H reactions by any H complexes within prothrombinase, but may antagonize inhibition by the conjugate through unproductive interactions of ATH with uncovered sites close to Xa at the phospholipid surface. These findings reveal important features involved in the enhanced anticoagulant action of ATH against prothrombinase surface-bound enzymes. Disclosures:No relevant conflicts of interest to declare.

Covalent Antithrombin-Heparin Complex Catalysis of Activated Protein C Inhibition by Protein C Inhibitor.

Abstract 3167Poster Board III-107 IntroductionThrombin (IIa), when bound to thrombomodulin (TM), readily converts protein C (PC) into activated PC (APC). APC functions as an anticoagulant by inactivating activated factors V (FVa) and VIII (FVIIIa), and indirectly reduces IIa generation. Once formed, APC activity is controlled through inhibition by PC inhibitor (PCI), a reaction that is catalyzed by heparin (H). Chan et al. developed a covalent antithrombin-heparin (ATH) complex with increased anticoagulant activity compared to H. The current investigation looked at the role and mechanism by which ATH affects the inhibition of APC by PCI. MethodsDiscontinous second order rate constant inhibition assays of APC+PCI, in the presence of ATH or unfractionated H (UFH), were performed. These experiments were repeated in the presence of low molecular weight H (LMWH) or ATH (LMWATH), or a high molecular weight ATH (HMWATH). The affinity of ATH or UFH for APC or PCI was assessed using native electrophoresis and APC or PCI immobilized onto agarose beads. ResultsSecond order rate experiments revealed that, at peak values, ATH (k2=2.0 × 107 ± 1.2 × 106) was a significantly slower catalyst of APC inhibition by PCI than UFH (k2= 3.0 × 107 ± 2.0 × 106; p=0.005). The peak reaction rate value for UFH occurred at a higher catalyst concentration (300nM) compared to ATH (60nM). LMWH was a poor catalyst of APC inhibition by PCI (k2= 4.2 × 105 ± 8.2 × 104). However, both LMWATH and HMWATH had high catalytic function (k2= 4.1 × 107 ± 2.1 × 106 and k2= 2.7 × 107 ± 4.8 × 105, respectively). Although UFH and ATH were able to bind to either APC or PCI at a pH of 7.3, increasing the pH to 8.8 eliminated all binding affinity, except that of ATH for APC. ConclusionUFH functions mechanistically through a template-mediated effect by bridging APC with PCI. The catalytic function of UFH is dependent upon H chain length, as LMWH was ineffective at enhancing APC inhibition by PCI. On the other hand, ATH may act mainly through conformational APC activation. There was no H chain length dependence for ATH, as LMWATH was able to significantly catalyze PCI inhibition of APC. We speculate that ATH's AT moiety may assist interaction with APC but prevents the H component of ATH from bridging the enzyme with the inhibitor. Compared to UFH, ATH is a more potent inhibitor of IIa, but is less effective at inhibiting APC and thereby decreasing IIa generation. These results add to the growing evidence that ATH is a superior anticoagulant to UFH. DisclosuresNo relevant conflicts of interest to declare.

Effect of covalent antithrombin-heparin complex on developmental mechanisms in the lung

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.

Review on Patents for Potent Anticoagulant Antithrombin-Heparin Covalent Complexes that Control Thrombosis In Vivo

Fractionated and low molecular weight heparins comprise the main class of anticoagulants used to prevent and treat thrombosis in patients through catalysis of enzyme inhibition by plasma antithrombin. However, clinical application of heparin suffers several limitations including: reduced venous half-life, uncontrolled anticoagulant effects, hemorrhagic complications and biophysical problems. A novel covalent antithrombin-heparin (ATH) complex has recently been created that overcomes heparins deficiencies. Historical patent literature and prior art reveal a progression of earlier attempts to link heparin to antithrombin that yielded products with various degrees of functionality. Relative to heparin, the present ATH invention of matter has a vastly increased bioavailability of circulating complex, significantly increased antithrombotic activity and superior ability to neutralize clot-bound coagulation enzymes. Recently issued ATH patents describe ATH-coatings on surfaces of devices that come into contact with blood. These ATH-covered products are vastly improved in prevention of device malfunction due to thrombus formation. There is a large market related to the biomaterial coatings industry that may benefit from development of blood-contacting products containing surface-bound ATH. Keywords: Antithrombin, heparin, antithrombin-heparin complex, thrombin, anticoagulant, antithrombotic, thrombosis, catalysis, plasma, coating

Protein adsorption on polyurethane catheters modified with a novel antithrombin‐heparin covalent complex

Highly anticoagulant covalent antithrombin-heparin complex (ATH) was covalently grafted onto polyurethane catheters to suppress adsorption/activation of procoagulant proteins and enhance adsorption/activation of anticoagulant proteins for blood compatibility. Consistency of catheter coating was demonstrated using immunohistochemical visualization of ATH. The ability of the resulting immobilized ATH heparin chains to bind antithrombin (AT) from plasma, as measured by binding of (125)I-radiolabeled AT, was greater than that for commercially-available heparin-coated catheters, and much greater than for uncoated catheters. Complementary measurements of antifactor Xa (FXa) activity and plasma protein binding were also performed. Both ATH-coated and heparin-coated catheters demonstrated functional binding of exogenous AT. However, the ATH-coated catheters gave a trend towards elevated anti- FXa activities/AT binding ratios, consistent with the higher active pentasaccharide content in starting ATH. Western blot analysis of proteins adsorbed to catheters after incubation with rabbit plasma established protein binding profiles that showed AT and albumin as major plasma proteins adsorbed to ATH-coated catheters, while AT and altered forms of fibrinogen were major plasma protein species adsorbed to heparinized catheters.

Biodistribution of covalent antithrombin-heparin complexes

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.

Reaction of Fibrin-Bound Thrombin with a Covalent Antithrombin-Heparin Complex Results in an Anticoagulant Surface.

Thrombin bound to fibrin is protected from inhibition by mixtures of antithrombin (AT) and heparin (H). Resistance of thrombin to reaction with AT + H when fibrin is present results from the formation of a ternary fibrin:thrombin:H complex. However, recent investigations using an antithrombin-heparin conjugate (ATH) showed that the covalent ATH complex rapidly inhibits fibrin-bound thrombin, with the thrombin-ATH product remaining attached to the fibrin. One intriguing possibility from these findings was that the heparin chain of ATH (or thrombin-ATH) on the fibrin surface might still be available to interact with fluid-phase molecules. We studied the anticoagulant activity of fibrin-bound ATH by determining the ability to catalyze the inhibition of added thrombin + AT. Excess fibrin monomer (polymerization inhibited by GPRP) was mixed with thrombin to form soluble complexes. Fibrin:thrombin was then incubated in the absence or presence of AT, H, AT + H or ATH, at various concentrations relative to the thrombin. Following treatment with anticoagulant, a vast excess of exogenous thrombin + AT was added and the ability of fibrin:thrombin-bound heparin to catalyze inhibition of the exogenous thrombin was assessed by measuring the loss of thrombin activity (chromogenic substrate assay). In the absence of heparin, reaction of exogenous thrombin + AT was essentially unaffected by the presence of soluble fibrin:thrombin. When fibrin:thrombin complex was mixed with AT + H (concentrations equimolar to thrombin), an insignificant ability to catalyze inhibition of excess thrombin by AT was observed (remaining thrombin activity = 49 +/− 4 vs 43 +/− 3 mOD/min for reaction with AT + H vs without AT + H, respectively). However, when similar concentrations of ATH were combined with fibrin:thrombin, a marked catalytic activity was seen (remaining thrombin = 34 +/− 3 mOD/min). Furthermore, the significant catalytic effect of ATH could be demonstrated over a wide range of concentrations relative to fibrin:thrombin. At these same concentrations, AT + H had little effect on exogenous thrombin + AT reactions. We conclude that although AT + H is unable to effectively inhibit fibrin-bound thrombin, covalently linked ATH not only overcomes this defect but can convert the fibrin:thrombin into a new site for anticoagulant activity to catalyze thrombin inhibition by exogenous AT. If clot-based antithrombin activity is demonstrated in vivo, we speculate that ATH may be useful for transforming thrombi from procoagulant to anticoagulant entities.

Isoform composition of antithrombin in a covalent antithrombin–heparin complex

Antithrombin (AT) circulates in two isoforms, α- (90–95%) and β-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% α-AT, and 45% β-AT) was significantly different than that in the commercial AT starting material (80% α-AT and 20% β-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the β-variant reacted ∼2-fold faster.