• anticoagulant;
  • antithrombin;
  • heparin;
  • polyurethane;
  • surface grafting


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

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 125I-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. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2007


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

Thrombus formation and bacterial infection are major, unresolved problems associated with blood contacting devices such as catheters, grafts, and artificial hearts.1–3 Surface modification is commonly used to make the materials more blood-compatible, while minimizing any loss of mechanical properties. Two approaches to modification have been commonly used. Suppression of nonspecific protein adsorption4 using coatings of polyethylene oxide (PEO) (a neutral, hydrophilic, and highly flexible polymer)5 or other hydrophilic polymers has been investigated for surface passivation.6 Uncontrolled, nonspecific protein adsorption, which usually occurs within seconds following the exposure of a foreign surface to blood, can initiate blood coagulation and the complement pathways.7 A second approach has been to use coatings that actively assist the anticoagulant activity of surfaces. Certain plasma proteins (such as antithrombin (AT) which can inhibit thrombin and factor Xa (FXa))8, 9 or heparin10, 11 (a glycosaminoglycan which catalyzes the reactions of plasma AT) have been used for this purpose.12, 13 The combination of PEO14–24 and heparin on the same surface11, 25 has been shown to improve blood compatibility compared to either component alone26–28 for an FDA-approved oxygenator and other clinical applications.29, 30

Heparinized surfaces are prone to leaching, nonuniform distribution, and variable anticoagulant activity. In addition, inactive, highly negatively-charged heparin chains may promote the binding of proteins that may increase prothrombotic activity.11 A new active agent, antithrombin-heparin covalent complex (ATH), has been recently developed that does not have the drawbacks of heparin. ATH was found to have very high anticoagulant activity,31 probably due to the fact that all of the ATH molecules have the pentasaccharide sequence essential to activate AT and that half of the ATH molecules have two active pentasaccharide sequences.31–33 By contrast, only one-third of the molecules in standard unfractionated heparin (UFH) preparations have pentasaccharide sequences.31, 32 Enhanced pentasaccharide content in ATH occurs during synthesis from selective binding of only high affinity pentasaccharide containing heparin by the AT, followed by a spontaneous Schiff base- Amadori rearrangement reaction between AT amino groups and heparin aldose termini.31, 34 In addition, the covalent binding of AT to heparin in ATH has been shown to give reduced nonspecific binding of other plasma proteins compared to UFH.35 Surfaces coated with ATH may thus have significantly improved anticoagulant properties.

The primary objective of the present report was to investigate mechanisms that may be involved in the increased anticoagulant and biocompatible functions in vivo of ATH-coated catheter surfaces. Since our previous report showed ATH-coated catheters to have better in vivo patency than that of commercial heparin-coated and noncoated catheters,36 tests with these devices were also included for a general comparison. To pursue mechanistic study, the capacity of the coatings to bind (using 125I-labeled AT) and activate (as in anti-FXa heparin activity assays) AT, as well as the capability to adsorb pro- or anticoagulant molecules from plasma, was determined. The test surface reported in this communication consisted of a protein repellent polyurethane to which ATH is attached as a coating by either covalent or noncovalent means. The use of an intermediate basecoat provides a procedural platform that allows coating to different substrates with only minor changes in basecoat chemistry. PEO was used as a spacer for ATH attachment, and as an auxiliary basecoat-blocking agent to suppress nonspecific adsorption of proteins. Evidence of coverage of the coated surface was evaluated using immunohistograms. Specific anticoagulation effects of the modified surface were investigated using AT adsorption and anti-FXa assays. Protein adsorption was studied by immunoblotting after plasma exposure.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References


Unless otherwise stated, chemicals used were of analytical grade. Human plasma AT was obtained from Bayer (Mississauga, ON, Canada) and UFH was grade I-A standard heparin (Na salt from porcine intestinal mucosa, 209 U/mg) from Sigma (Oakville, ON, Canada). ATH was prepared by incubation of AT and UFH in PBS at 40°C for 14 days, with subsequent purification on butyl-agarose (to remove unreacted heparin) and DEAE Sepharose (to remove unreacted AT), as described previously.31125I-labeled AT was prepared by incubating AT with Na125I (New England Nuclear, Mississauga, ON, Canada) and Iodobeads (Pierce, Mississauga, ON, Canada).37 Thrombin and FXa were both from Enzyme Research Laboratories (South Bend, IN). N-Hydroxysuccinimide (NHS)-PEO (3400)-acrylate and PEO-methacrylate were purchased from Shearwater Polymers (Huntsville, AL). Ethylene glycol dimethacrylate, diurethane dimethacrylate, PEO methacrylate, methyl methacrylate, urethane, dichloromethane, 2,2′-azobis(isobutyronitrile) (AIBN), and SDS-polyacrylamide gel electrophoresis (PAGE) chemicals were purchased from Aldrich (Oakville, ON, Canada). Gel staining agents (Alcian Blue and Coomassie Blue) and protease (P-5147, from Streptomyces griseus) were from Sigma. Agents for bioactivity measurement (anti-FXa heparin activity assay kits) were purchased from Diagnostica Stago (Asnieres, France). Chromogenic substrate for thrombin, S-2238, was purchased from DiaPharma (Milan, Italy). Uncoated polyurethane catheters (PU-C70) and heparin-coated polyurethane catheters (CBAS-C70) from Solomon Scientific (Plymouth Meeting, PA) were 7 French gauge with rounded tips and were 15 cm in length. Fluorescein and PD-10 columns were purchased from Pierce (Rockford, IL). Antibodies against human AT, albumin, and thrombin were purchased from Affinity Biologicals (Hamilton, ON, Canada). Antibodies against human prekallikrein, plasminogen, α-2-macroglobulin, heparin cofactor II, and vitronectin were purchased from Cedarlane Laboratories (Hornby, ON, Canada).

Preparation of fluorescently-labeled rabbit anti-sheep IgG

Rabbit antisheep IgG in 0.1M sodium carbonate, pH 9.0, was allowed to react with fluorescein 6-carboxyl propionic acid N-succinimidyl ester (1 mg/mL in DMF) for 2 h at 4°C. The labeled antibody was then separated from unbound fluorescene on a PD-10 column. Fractions were collected and monitored using a fluorometer (SpectraMax Gemini spectrometer, from Molecular Devices, Sunnyvale, CA, with filter settings at excitation 485 nm and emission at 555 nm).

Description of commercial catheters

Polyurethane (PU-C70) catheters from Solomon were composed of nonhalogenated polyurethane from a combination of polyurethanes coded 80 A and 55 D.38 Although exact composition of repeating polymer blocks and chain extenders is not specified, the main urethane structure ([BOND]O[BOND]CO[BOND]NH[BOND]) results from an ∼1:1 reaction of polyols (such as polytetramethylene ranging in molecular weight from 500 to 3000) and small diisocyanate monomers.38, 39 Heparin-coated polyurethane (CBAS-C70) catheters were commercially prepared by introduction of an amine-rich polyurethane–urea onto the catheter surface, followed by covalent bonding of an aldehyde-terminating heparin fragment by covalent end group attachment to the amines on the coating.39 In brief, prepolymers were prepared by heating polyols (diol molecular weight varying from 400 to 3000) with diisocyanates in appropriate solvents. Prepolymer solution was added to alkyl-diamine solution at 30°C to give the amine rich polyurethane–urea, which was then flow- or dip-coated onto the polyurethane catheter. Heparin fragments with aldehyde-containing anhydromannose end groups (generated by treatment of UFH with HNO2) were then reacted with the amine rich coating to form a Schiff base with the aldehyde end groups, the resultant imines being converted to stable secondary amines by reduction with NaBH3CN.39, 40

Covalent coating of catheters with ATH

Coating of catheters with ATH followed the following strategy. ATH was reacted with a heterobifunctional reagent containing an NHS end group that became attached via amine groups on the AT portion of the ATH. The derivatized ATH was then allowed to copolymerize (via acrylate groups on the other end of the bifunctional reagent) with double bonded reagents dried onto the catheter surface (double bonds activated with AIBN). ATH (60 mL of 4.0 mg (AT)/mL) was reacted with NHS-PEO(3400)-acrylate (0.703 g) to form an activated ATH–PEO complex (dialyzed versus H2O prior to use). Polyurethane catheters were then modified with ATH–PEO as follows. A basecoat was applied to polyurethane catheters by dipping them into a solution of 1.5 g (0.015 mol) methyl methacrylate, 4.5 g (0.023 mol) ethylene glycol dimethacrylate, 1.5 g (0.0032 mol) diurethane dimethacrylate, 4.5 g (0.00045 mol) PEO methacrylate, and 0.03 g (0.00018 mol) AIBN in 55 mL dichloromethane, and allowing incubation to proceed for 10 min. The catheters were removed from the base-coating solution tube and dried in a vacuum chamber. The base-coated catheters were then reacted with ATH–PEO (60 mL of 4.0 mg (AT)/mL) in the presence of Triton X-114 (0.05 g added) at 80°C (2 h), followed by an annealing step at 50°C (1 h). Catheters were then washed three times with saline. To confirm that AIBN was intact on the catheter surface to launch grafting of the ATH–PEO, a few experiments were conducted with ATH–PEO in the presence of an antioxidant radical-quencher (ascorbate) and it was observed that no anticoagulant ATH became bound to the surface. This would suggest that when no quencher is present, AIBN generation of radicals is occurring.

Confirmation of surface coverage by coating

The low-level homogeneity of ATH on coated catheters was tested using immunohistochemical methods. ATH-coated catheters were washed with deionized water and incubated with 1.0 mL 5% powdered skim milk in Tris-buffered saline (TBS, 0.05M Tris.HCl, 0.1M NaCl, pH 7.4) for 1 h, followed by three washes with 0.1% skim milk in TBS. The catheters were then incubated for 1 h with sheep antihuman AT antibody in 1 mL 1% skim milk-TBS containing 0.5% (w/v) Tween 80 (TBST). After reaction with the primary antibody, the catheters were washed four times with 0.1% skim milk in TBST, and reacted with the fluorescently labeled rabbit antisheep IgG in TBST for 1 h. The tubing was washed again with TBST and tested for fluorescence, using an Axioskop 2 microscope equipped with high performance epi-fluorescence equipment. The excitation filter was set at 450–490 nm, and the emission filter was set at 505–590 nm. Uncoated control catheters were processed in parallel for comparison.

Characterization of AT binding to catheters

Adsorption of 125I-AT to the surface of catheters was used to evaluate heparin-AT binding activity. The protein solutions for adsorption studies (up to 0.15 mg protein/mL (typical plasma concentrations)) consisted of 98% unlabeled and 2% radiolabeled AT in phosphate buffered saline (PBS, 0.01M sodium phosphate, 0.14M NaCl, pH 7.4). Catheters were incubated with 250 μL 125I-AT solution (0.01–0.15 mg/mL) for 2 h at room temperature, rinsed three times in PBS, wicked onto filter paper (to remove residual adherent buffer), and the radioactivity of the catheters measured. A minimum of four replicates were measured for each type of catheter studied.

Adsorption of AT from plasma was also measured. It was expected that adsorption from plasma would be predominantly via specific interactions with surface-bound heparin due to blockage by plasma proteins of nonspecific binding. For these experiments, radiolabeled AT was added to plasma in amounts corresponding to 2% of the native AT content in plasma (3 μg/mL). The procedure was the same as for the buffer experiments.

Gel and immunoblot analysis

Reduced SDS-PAGE (12% gels) was used to separate proteins eluted from catheter segments incubated with rabbit plasma. The 0.5-cm catheter segments were incubated for 2 h at 23°C in pooled normal plasma obtained from rabbits and anticoagulated with 0.32% (m/m) sodium citrate. The catheter segments were then rinsed with normal saline and incubated overnight with 2% SDS at 23°C to elute adsorbed proteins. Five microliter samples of each eluate were added to denaturing sample buffer and heated at 90°C for 5 min. SDS-PAGE was performed following protocols detailed in previous reports.41 Immunoblot analysis was then carried out as described before.41 Briefly, gels were blotted onto Immobilon polyvinylidene fluoride (PVDF) membranes (Millipore, Nepean, ON, Canada), and the blots cut into 3-mm wide strips. The strips were separately incubated with primary antibodies against individual rabbit plasma proteins, followed by alkaline phosphatase-conjugated anti-IgG. Bands were then visualized by incubation with NBT/BCIP substrate (Pierce, Rockford, IL). Initial plasma samples, as well eluates, were examined.

Anti-FXa assay

The ability to catalyze FXa inhibition by AT is a measure of the pentasaccharide content of heparin molecules since the reaction is directly dependent on conversion of AT into its active conformation upon binding to this specific sequence and structure.27 Surface-immobilized heparin was tested for anti-FXa activity using an amidolytic method with chromogenic substrate according to Teien et al.42 The method was modified slightly from the Diagnostica Stago (Asnieres-sur-Seine, France) protocol using the Stachrom® heparin kit. Catheter segments were incubated in 96-well microtitre plates with an excess of AT for 4 min at 37°C. Excess FXa was then added and allowed to react for 5 min at 37°C. A subsample of solution was then removed and incubated with the chromogenic substrate, CBS 31.39, for 1 min to measure residual FXa not inhibited by the AT. The reaction was stopped with addition of 50% acetic acid. Released p-nitroaniline was measured by absorbance at 405 nm. Increase in FXa inhibited by AT over the 5-min reaction period was a measure of the catalytic capability of heparin on the surfaces. The amount of active heparin on the original catheter could be calculated by reference to a heparin standard curve. All results were given in terms of pmol equivalents of standard heparin that gave the same FXa inhibitory activity and calculated per square centimeter of catheter surface area. Surface-immobilized heparin anti-FXa activity was measured for catheters prior to incubation with rabbit plasma as described previously.

Statistical analysis

Significant differences in groups of data were determined using analysis of variance (ANOVA). For comparisons between two groups, students t-test was used. Differences were considered significant for p < 0.05. Results were reported as mean ± SD.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

Coverage of catheter surface by ATH coating

The extent of coverage of coatings on the surfaces was investigated using anti-AT immunofluorescent histochemistry (Fig. 1) for ATH-coated catheters, base-coated catheters (no ATH), and uncoated control catheters. It was observed that the fluorescence intensity on the ATH-coated surfaces was much greater than that on the other two surfaces, indicating the presence of ATH with accessible AT on the surface. Uniformity of fluorescence at 80× magnification suggested that the coating was consistent over areas the size of a platelet or greater. This finding is consistent with the high resistance of ATH-coated catheters and endoluminal grafts to formation of even microthrombi in vivo.36, 43 It was possible, however, that coating heterogeneity may exist at the sub micrometer level.

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Figure 1. Immunohistogram of catheters treated with sheep antihuman AT and rabbit antisheep IgG conjugated with fluorescein. The excitation filter was 450–490 nm and emission was 505–590 nm. Left, antithrombin-heparin (ATH)-coated catheter; middle, basecoat-coated catheter; right, uncoated control catheter. Magnification, ×80.

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AT adsorption

Figure 2 shows isotherms for AT adsorption from buffer to modified and unmodified polyurethanes. Heparin-modified catheters (CBAS) were used as a commercial heparinized surface for comparison with the heparin-containing ATH-coated catheters. Adsorption increased with increasing protein concentration and saturation was not reached at the highest concentration used (0.15 mg/mL). Adsorption levels were substantial for all three surfaces, with the heparin-coated surface showing the highest adsorption, followed by the unmodified polyurethane (commercial control for comparison to the commercial heparin-coated catheters) and the ATH surface, which showed the lowest adsorption. At the highest concentration, binding ranged from 6 to 9 pmol/cm2. For a protein of MW ∼60,000 Da, these amounts are in the order of those expected for monolayers.

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Figure 2. Isotherms for AT adsorption from buffer to modified and unmodified polyurethanes (PU) at room temperature for 2 h. The heparin-modified surface was used as a reference. PU-ATH, polyurethane-antithrombin-heparin covalent complex surface; PU, uncoated polyurethane surface; PU-H, polyurethane-heparin surface. Values are mean ± SD (n = 3).

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Adsorption of AT from plasma was expected to show differences between the heparinized and nonheparinized surfaces because of the relatively high affinity of AT-heparin interactions. The other much more abundant plasma proteins were expected to reduce low affinity binding (similar to blocking of low affinity binding in ELISAs by albumin), as opposed to the high affinity AT-activation sites on heparins. To exhibit surface activity, the heparin molecules must be in an orientation that favors their contact with fluid-phase AT.44–48 Thus, any increase in binding of AT on heparin or ATH surfaces may also be an indication of the functional availability the heparin moiety on the surface.

Figure 3 shows AT adsorption from plasma. The polyurethane (PU)–ATH surface showed the highest adsorption, reflecting availability of greatest number of high AT-affinity sites. The heparinized surface also showed significant AT adsorption from plasma, while the unmodified PU surface showed a value close to background. Comparison of Figures 2 and 3 shows that when physiological concentrations of plasma protein was present, while total binding of AT to all surfaces was reduced, (PU)–ATH showed a significant trend towards greater AT-binding relative to uncoated surfaces (p < 0.05, Fig. 3). Although binding of AT from a plasma-milieu by heparin-coated surfaces was measurable, the amount was not statistically different from that of uncoated PU.

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Figure 3. Adsorption of AT from plasma to polyurethane-based surfaces (room temperature, 2 h). PU-ATH, polyurethane-antithrombin-heparin covalent complex surface; PU-H, polyurethane-heparin surface; PU, uncoated polyurethane surface. Values are mean ± SD (n = 3). * denotes a significant difference (p < 0.05) in AT binding compared to the PU control surface.

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SDS-PAGE and Western blots

As a complement to the measurement of adsorption from plasma using radiolabeled AT, SDS-PAGE and immunoblotting analysis were performed on the proteins eluted from coated catheters after contact with rabbit plasma. The data from these experiments provided a more comprehensive picture of the overall composition of the protein layer deposited onto the surfaces. It should be noted that for these experiments: identical catheter areas were used for all analyses, identical volumes of elutant were used, and identical volumes of eluate were loaded on the gels. Thus, the intensity of the bands on the gels and blots gave an indication of the relative quantities of the proteins adsorbed to the different surfaces. However, interpretation of relative protein binding between the commercial heparin surface and ATH–PEO surface is confounded by the potential difference in heparin chains, amounts of surface-bound heparin, base coats, and PEO linker. Thus, the discussion below is guarded on comparisons between catheter types.

Some inherent difficulties that exist regarding the semiquantitative analysis of eluted proteins on SDS PAGE/ Western immunoblots should be noted. For example, high molecular weight and less polar molecules may have slightly reduced yields as more points of detergent contact are required for suspension.49 Thus, a micellular interaction or entrapment mechanism may be involved for removal of these proteins. In fact, for some strongly hydrophobic surface–protein interactions (such as for membrane proteins), a combination of chaotropic agents + detergents has been commonly used,50–52 as well as organic solvents in some cases.53, 54 Nevertheless, SDS is a strong ionic, amphiphobic detergent that has been recommended as one of the most efficient surfactants52 and is commonly used for plasma protein solubilization prior to immunoblotting.55 In fact, as in the present study, we have previously reported data that carefully detailed significant differences between newborn and adult plasma protein binding to a variety of surfaces using this method.41 Polyacrylamide gels of ATH (or heparin) mixed with plasma, followed by buffered SDS at room temperature, show no binding of Coomassie stained proteins to the ATH or heparin. Furthermore, as a proof that washing with SDS is complete, repeat SDS washings of the SDS-treated surfaces did not remove any further detectable protein. Still, variation of elution conditions, detergents, or solvents might theoretically have helped to remove any small amounts of surface-bound protein still remaining. Although analysis by elution and immunoblotting might not be preferred to direct antibody detection on the catheter surface, design and standardization of a direct method is difficult. However, we are in the midst of developing such a technique56 which will hopefully confirm results reported here.

It appeared from the blots shown in Figure 4 that the heparinized and uncoated polyurethane control catheters adsorbed greater amounts of protein than the catheter coated with ATH–PEO (note: right lanes without immunodevelopment, where total protein profiles are shown). Thus, the ATH–PEO surface may have reduced overall affinity for plasma proteins (densitometric analysis of total protein band staining indicated that the ATH–PEO-coating adsorbed 36% and 31% of the amount of protein taken up on heparin-coated and uncoated surfaces, respectively). Antibodies against prekallikrein, fibrinogen, plasminogen, AT, albumin, α-2-macroglobulin, thrombin, heparin cofactor II, and vitronectin were used in the blotting experiments. In general, the protein deposition patterns were complex, and most of the proteins probed for were found to be present on all three materials. However, some differences were observed in the types of proteins bound by the three surfaces, for example, no AT was detected on the uncoated control. Also, fragments of certain proteins were variously detected, for example, fibrin(ogen) degradation products were observed on uncoated and heparin-coated surfaces.

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Figure 4. Western blots of proteins eluted from the polyurethane (PU) catheters, heparin-coated catheters (CBAS), and ATH-coated catheters after contact with plasma. Reduced SDS-PAGE (12% gels) was used to separate proteins eluted from catheter segments incubated with rabbit plasma, blotted onto membranes, and the blots cut into 3-mm wide strips. The strips were separately incubated with primary antibodies against individual rabbit plasma proteins, followed by alkaline phosphatase-conjugated anti-IgG. Bands were then visualized by incubation with NBT/BCIP substrate. The lanes at the extreme left in each of the three blots are molecular weight (MW) standards while the lanes to the right are gold-stained SDS-PAGE gels of the eluates.

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The behavior of AT was of particular interest since the anticoagulant activity of heparin is mediated by this protein. Both the ATH-coated catheter and the heparinized catheter showed significant adsorption of AT (Fig. 4, circled regions). However, no AT was detected on the uncoated control catheter. Multiple bands were seen in the AT region for the heparinized catheter, possibly due to the well-known heterogeneity in AT glycosylation.57 The ATH modified surfaces showed only a single AT band, suggesting that only one form of AT was adsorbed; the lower molecular weight of this band being suggestive of β-AT isoform, which has a higher affinity for heparin binding sites.57

The high molecular weight AT-containing band from the heparinized surface is typical of the 90 kDa thrombin–antithrombin (TAT) complex58–60 (generated either in vitro or in the animal prior to blood collection). The presence of TAT revealed that thrombin generation had taken place in a procoagulant environment. Since release of TAT complex is required for the continued catalytic function of the heparinized surface, retention of TAT on the heparinized surface (CBAS, Fig. 4) would limit the heparin's ability to catalyze further thrombin inhibition (hinder approach of more incoming AT to the surface). In the case of the ATH-coated surface, interactions with thrombin were complicated. As previously demonstrated,32, 33 two separate reactions can occur involving thrombin and ATH. The first reaction is the rapid direct inhibition of thrombin by attaching covalently to the AT moiety in ATH to form an inert thrombin–ATH complex.31 This reaction occurs only once, since ATH can only form an irreversible complex with one thrombin molecule. Unfortunately, since the covalent thrombin–ATH complex cannot dissociate, the bound thrombin could not be eluted by buffer and observed on the Western blot (Fig. 4). The second reaction involves the fact that the heparin chain in either ATH or thrombin–ATH has the ability to bind AT from plasma and catalyze the reaction of that absorbed AT with incoming thrombin to form thrombin–AT (TAT).32, 33 Once formed, TAT can dissociate from the ATH heparin so that the heparin chain is free to bind and activate more plasma AT for reaction with thrombin. Evidence for both direct and catalytic inhibition of thrombin by ATH has been shown with ATH-coated grafts43 and proof that the plasma AT-activating reaction can occur was given in the present study by the fact that catalytic anti-Xa activity was measured on the ATH catheters (Table I, see below).

Table I. Antifactor Xa (FXa) Activity and Antithrombin (AT) Adsorption on Different Polyurethane Catheters
 Anti-Xa Activity (pmol/cm2)AT Adsorption from Buffer (pmol/cm2)Ratio (Activity/Adsorption)
  • PU-control, polyurethane control catheters; PU-heparin, polyurethane heparin-coated catheters; PU-ATH, polyurethane antithrombin-heparin covalent complex-coated catheters.

  • Values are mean ± SD.

  • *

    p < 0.05 denotes significant difference relative to the PU-control.

PU-Control0.007.68 ± 1.770
PU-Heparin8.42 ± 10.00*9.55 ± 2.060.91 ± 0.20*
PU-ATH10.36 ± 5.87*6.37 ± 0.84*1.65 ± 0.22*

Fibrinogen (molecular weight, 340 kDa) plays a critical role in the formation of fibrin clots. It is converted to fibrin monomer by the action of thrombin, which then polymerizes to form a fibrin clot. Intense bands of intact fibrinogen, as well as fibrin(ogen) fragments, were found on the unmodified control and heparinized catheters, suggesting that both fibrinogen deposition and denaturation/degradation (thrombin activity) had occurred on those surfaces. Relatively small amounts of intact fibrinogen and fibrin(ogen) fragments were present on the ATH-coated catheter, suggesting a reduced surface procoagulant activity (thrombin generation). A potential reason for coagulant activity on the heparin surface is that, since partial depolymerization of heparin is required to get active end groups for heparin coating,39 some chains may be too short to simultaneously bind both the AT and thrombin to neutralize thrombin activity via a bridging mechanism.61

Anti-FXa activity

Data on anti-FXa activity for the surfaces studied are listed in Table I. Both the polyurethane heparin-coated (PU-H) and the polyurethane ATH-coated (PU-ATH) materials showed significant anti-FXa activity, reflecting the presence of functional heparin on these surfaces. The control polyurethane material (PU-control) showed no activity. Previous characterization of the ATH-coated and heparin-coated catheters using periodate has verified that the anticoagulant activity in ATH-coated and heparin-coated catheters is entirely due to the pentasaccharide sequence.36 In the case of ATH, exogenous AT can noncovalently bind (and be activated by) the heparin pentasaccharide site on the heparin chains of ATH, even though the conjugated AT in ATH remains covalently attached to the aldose terminus of the heparin moiety.33 Thus, although FXa reacts to produce a FXa-ATH inhibitor complex (or thrombin-ATH in the case of thrombin assays), the ATH heparin chain still retains the ability to potently catalyze reaction of more FXa with exogenous AT.31, 33 The rationale for analysis in the activity assay relies on the validity for use of the standard heparin in the kit as a reference. ATH is a covalent 1 mol:1 mol complex of AT and heparin,31, 33 where the catalytic anti-FXa activity resides only in the heparin moiety31 (thus making it justifiable to refer to standard heparin in the kit). Consequently, because the assay measures the amount of heparin-like activity in the sample that catalyzes inhibition of FXa by added AT, the activity could be calculated as the pmol of active surface-bound ATH in terms of equivalent pmol of standard heparin from the kit. In the case of PU-heparin, a similar calculation was done to get pmol of standard heparin anti-FXa activity on the surface.

Anti-FXa activity and AT adsorption, as determined in this study, provide measures of different aspects of heparin activity. The assay used for anti-FXa activity on a surface measures the number of heparin sequences that can activate AT to give a conformation which reacts rapidly with FXa.62 AT adsorption from plasma measures the number of AT molecules that interact with a minimum level of affinity for the surface (especially found for pentasaccharide-containing heparin molecules).63, 64 Therefore, it is of interest to compare anti-FXa activity and AT adsorption. The anti-FXa assay gave the “turnover” rate of heparin related to the catalysis of inhibition by bound AT. For the heparin-modified surface, the anti-FXa assay showed a value similar to the AT adsorption (Table I). For the ATH-modified surface, the anti-FXa assay gave a higher value than the AT adsorption, a result significantly different to that of the heparin-coated surface (p = 0.023 (2-sample students t-test) for activity/adsorption ratios of PU-heparin versus PU–ATH in Table I). With respect to AT adsorption, it is highly likely that adsorbed AT will remain bound if no further interactions occur with either FXa or thrombin.64 In that case, the maximum quantity of AT that could be adsorbed would simply be that of a monolayer (assuming the binding site density is sufficient). To exhibit anti-FXa activity, however, the bound AT must be converted to its activated conformation, interact with FXa, and the resultant inhibitor complex must be released from the surface.27 If some molecules on the surface could bind AT but not effectively activate the AT to inhibit FXa, compared to the reference heparin, then the ratio of pmol of surface heparin that could inhibit FXa (by AT) to the total pmol of AT that the surface can absorb would be less than 1. In the case that the ratio of pmol of active heparin that catalyze FXa inhibition to pmol of AT bound is greater than 1 (as with the ATH surface), one implication might be that the surface contains molecules that can activate a higher proportion of adsorbed AT for FXa inhibition than the heparin used as a standard. Such a finding was not surprising for ATH since, during synthesis of the conjugate, AT molecules select for heparin chains with the highest AT-affinity and pentasaccharide number to generate the anticoagulant complex.31, 32

Several factors may account for the decreased ratio of anti-FXa activity/bound AT in PU-heparin compared to ATH surfaces. Only one-third of the heparin chains in heparin have active AT-binding sequences,65 whereas in ATH all heparin chains contain at least one pentasaccharide sequence for activation of AT molecules taken up from plasma.32 Furthermore, heparin in the CBAS catheters was covalently bound to an amine-rich coating on the polyurethane catheters via end-point attachment66 through aldehydes generated by partial depolymerization of UFH with nitrous acid.39 Thus, although high substitutions of heparin up to 90.7 ± 4.9 μg/cm2 has been obtained by such oxidative methods,66 AT-activating pentasaccharide sequences may have been degraded. However, as stated above, since PEO is used in the ATH but not the heparin coating and the commercial heparin and ATH surfaces have different base coats, many other potential explanations exist for variation in AT-binding site activities. To resolve this issue, it will be necessary to coat heparin and ATH to the same catheter type with the same linking agent.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

A surface consisting of ATH immobilized onto polyurethane was developed. Using immunohistochemistry, it was shown that ATH was distributed over much of the surface. AT adsorption studies and measurement of the profiles of proteins adsorbed from plasma indicated that the ATH-modified surface was able to significantly attract AT in the presence of plasma proteins, an important requirement for a heparinized surface to display anticoagulant properties. Immunoblotting studies showed that AT was detected on both the heparinized- and ATH–PEO-modified surfaces. However, the surface modified with ATH–PEO also showed lower binding of the fibrinogen and fibrin(ogen) de gradation products. Anti-FXa assays of the ATH-modified surfaces suggested that the heparin component of the coating was functional and useful for antithrombotic purposes. Analysis of results for anti-FXa activity and AT adsorption on ATH–PEO surfaces may indicate the potential for increased activation of bound exogenous AT. Thus, ATH appears to be a good candidate for coating cardiovascular devices, such as catheters, requiring significant long-term blood compatibility. Further experiments are ongoing to test the antithrombic or prothrombotic nature of these different surfaces in chronic animal models.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

We would like to thank Nethnapha Paredes for assistance in the preparation of this manuscript. A.K.C.C. holds a Career Investigator award from the Heart and Stroke Foundation of Canada.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References
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