Covalent linkage of streptokinase to recombinant hirudin: a novel thrombolytic agent with antithrombotic properties.

In a continuing effort to create an agent which has both thrombolytic and antithrombotic properties, streptokinase (SK) was covalently bound to the potent antithrombin agent recombinant hirudin (rHir). Linkage of SK to 125I-rHir was accomplished via heterobifunctional crosslinkers in an average molar ratio of 1:1. The 125I-rHir-SK complex was purified from starting components by anion exchange and gel filtration chromatography. The major band containing covalently bound 125I-rHir had a molecular weight of 53 kDa as determined by SDS-PAGE and autoradiography. Biologic activity of each component was then assayed utilizing the chromogenic substrate for each compound. Complex bound 125I-rHir exhibited a 1.2 fold decrease in thrombin inhibition when compared to concentrations of 125I-rHir greater than 3.13 nM. Complex bound 125I-SK, replacing the 125I label on rHir, displayed a 7.9-fold loss in plasminogen activation when compared to 125I-SK. These chromogenic assay results were not adversely altered in the presence of the converse compound's substrate. The 125I-SK-rHir complex (examined at various concentrations) also demonstrated a 0.17- to 17-fold greater affinity for thrombin immobilized onto Sepharose beads as compared to 125I-SK. These findings indicate the rHir-SK complex maintained both thrombolytic and antithrombin properties while also obtaining affinity for immobilized thrombin.

Covalent linkage of recombinant hirudin to poly(ethylene terephthalate) (Dacron): creation of a novel antithrombin surface.

Thrombus formation and intimal hyperplasia on the surface of implantable biomaterials such as poly(ethylene terepthalate) (Dacron) vascular grafts are major concerns when utilizing these materials in the clinical setting. Thrombin, a pivotal enzyme in the blood coagulation cascade primarily responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous strategies to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) to a modified Dacron surface and characterize the in vitro efficacy of thrombin inhibition by this novel biomaterial surface. Bovine serum albumin (BSA), which was selected as the "basecoat' protein, was reacted with various molar ratios of the cross-linker sulphosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulpho-SMCC; 1:5-1:50). These BSA-SMCC complexes were then covalently linked to sodium hydroxide-hydrolysed Dacron (HD) segments via the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Covalent linkage of these complexes to HD (HD-BSA-SMCC) was not affected by any of the sulpho-SMCC cross-linker ratios assayed. rHir, which was initially reacted with 2-iminothiolane hydrochloride (Traut's reagent) in order to create sulphydryl groups, was then covalently bound to these HD-BSA-SMCC surfaces (HD-BSA-SMCC-S-rHir). The 1:50 (BSA: sulpho-SMCC) HD-BSA-SMCC-S-rHir segments bound 22-fold more rHir (111 ng per mg Dacron) compared to control segments and also possessed the greatest thrombin inhibition of the segments evaluated using a chromogenic substrate assay for thrombin. Further characterization of the HD-BSA-SMCC-S-rHir segments demonstrated that maximum thrombin inhibition was 20.43 NIHU, 14.6-fold greater inhibition than control segments (1.4 NIHU). Thrombin inhibition results were confirmed by 125I-thrombin binding experiments, which demonstrated that the 1:50 HD-BSA-SMCC-S-rHir segments had significantly greater specific thrombin adhesion compared to control segments. Non-specific 125I-thrombin binding to and release from the 1:50 HD-BSA-SMCC-S-rHir segments was also significantly less than the control segments. Thus, these results demonstrate that rHir can be covalently bound to a clinically utilized biomaterial (Dacron) while still maintaining its ability to bind and inhibit thrombin.

Coating of Dacron vascular grafts with an ionic polyurethane: a novel sealant with protein binding properties.

The purpose of this study was to develop a novel sealant that would seal prosthetic vascular graft interstices and be accessible for protein binding. Crimped knitted Dacron vascular grafts were cleaned (CNTRL) and hydrolyzed in boiling sodium hydroxide (HYD). These HYD grafts were sealed using an 11% solids solution of a polyether-based urethane with carboxylic acid groups (PEU-D) via a novel technique that employs both trans-wall and luminal perfusion. Carboxylic acid content, determined via methylene blue dye uptake, was 2.3- and 4.2-fold greater in PEU-D segments (1.0+/-0.27 nmol/mg) as compared to HYD and CNTRL segments, respectively. Water permeation through PEU-D graft (1.1+/-2 ml/cm2 min(-1)) was comparable to collagen-impregnated Dacron (9.8+/-10 ml/cm2 min(-1)). Non-specific 125I-albumin (125I-Alb) binding to PEU-D segments (18+/-3 ng/mg) was significantly lower than HYD and CNTRL segments. 125I-Alb linkage to PEU-D using the crosslinker EDC resulted in 5.7-fold greater binding (103+/-2 ng/mg) than non-specific PEU-D controls. However, covalent linkage of 125I-Alb to PEU-D was 4.9- and 5.9-fold less than CNTRL and HYD segments with EDC, respectively. Thus, ionic polyurethane can be applied to a pre-formed vascular graft, seal the interstices and create "anchor" sites for protein attachment.

In vivo assessment of a novel dacron surface with covalently bound recombinant hirudin.

Prosthetic arterial graft surfaces are relatively thrombogenic and fail to heal with a cellular neointima. The goal of this study was to characterize the in vivo antithrombin properties of a novel Dacron surface with covalently linked recombinant hirudin (rHir) implanted in a canine thoracic aorta with high flow and shear rates. rHir was bound to a knitted Dacron patch using crosslinker-modified bovine serum albumin (BSA) as a basecoat protein. BSA was first reacted with the heterobifunctional crosslinker, sulfo-SMCC. This BSA-SMCC complex was then bound to the carboxylic acid groups of hydrolyzed Dacron patches using the carbodiimide crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Iodinated, Traut's-modified rHir (125I-rHir-SH) was then reacted with the Dacron-BSA-SMCC surface, thereby covalently binding 125I-rHir. Graft segments were washed and sonicated to remove any nonspecifically bound 125I-rHir. Dacron-BSA-SMCC-S-125I-rHir patches (n = 5) and control Dacron-BSA patches (n = 5) were implanted in series in the thoracic aortas of canines. These patches were exposed to nonheparinized, arterial blood flow for 2 hours. Patches were explanted and assessed for 125I-rHir loss. Antithrombin activity of explanted 1-cm2 patch segments was evaluated using a chromogenic assay with 1, 5, 10, 15 units of added thrombin. Light microscopy was performed to qualitatively examine the pseudointima. Two animals were excluded from the study owing to excessive bleeding through the knitted 125I-rHir patch. Comparison of preoperative and postoperative 125I-rHir gamma counts revealed an overall decrease of 20+/-5.4% over the period studied. Explanted 125I-rHir patch segments were able to inhibit 1, 5, and 7 NIHU of thrombin, demonstrating retained antithrombin activity. Gross and microscopic examination of the control and test Dacron surfaces showed marked differences. Dacron surfaces with covalently bound 125I-rHir had no gross thrombus and a thin pseudointima of platelets and plasma proteins. In contrast, the control patches had a thick pseudointima composed of fibrin-rich thrombus. rHir, covalently bound to Dacron patches, maintains its biologic activity as well as prevents thrombus formation on the graft surface. This novel antithrombin coating, by modifying the blood/ graft interface, may improve both short- and long-term patency in small-diameter prosthetic arterial grafts and has applications with respect to other implantable or indwelling biomaterials.

Thrombin inhibition by covalently bound hirudin.

Hirudin is the most potent known natural inhibitor of thrombin and is presently gaining popularity as an anticoagulant since recombinant and synthesized forms have become available. We have made use of recombinant hirudin (rHir) by covalently binding it to both biomolecules and prosthetic biomaterials. Heterobifunctional crosslinking reagents were used to derivatize rHir and form covalent crosslinks between rHir and albumin producing active conjugates. Both derivatized rHir and conjugates inhibited human alpha-thrombin similarly, however, both showed a ten-fold decrease of alpha-thrombin inhibition when compared to rHir alone using the tripeptide substrate, S-2238. Immobilization of 2.75 +/- 0.45 micrograms rHir on 1.0 cm2 Dacron prosthetic graft patches resulted in inhibition of 1.88 +/- 0.03 micrograms alpha-thrombin in solution (mean +/- SD, n = 3), which is a 8:1 molar ratio, respectively. rHir ED50 inhibition of 0.1 NIH U alpha thrombin stimulated whole blood platelet aggregation was 0.12 x 10(-6) microM. The conjugate ED50 inhibition was 1.37 x 10(-6) microM showing an eleven-fold loss of activity. We conclude that there is only a ten-fold loss of inhibitory activity when rHir is covalently immobilized and that this technique has a benefit of localizing antithrombin activity to surfaces or soluble carrier molecules.

Synthesis and characterization of a recombinant hirudin-albumin complex.

The purpose of this investigation was to covalently bind recombinant hirudin (rHir) to albumin and compare alpha-thrombin inhibition by complexed rHir to rHir. rHir was radiolabelled with 125I and covalently bound to albumin using heterobifunctional cross-linking reagents. HPLC purification of the 125I-rHir-SMCC-albumin complex using gel filtration chromatography resulted in four elution peaks, with the main peak containing an average M(r) of 78 kDa. This peak fraction also contained 63% (+/- 1.4%) of the total protein and 49% (+/- 6.8%) of the 125I-rHir conjugated to albumin. Purification of unbound 125I-rHir from complex was confirmed by SDS gel electrophoresis and autoradiography. 125I-rHir inhibition of alpha-thrombin, measured by an assay utilizing the chromogenic tripeptide substrate H-D-Phe-Pip-Arg-pNA (S-2238), was observed to be non-competitive of linear mixed-type having a Ki of 1.61 pM and an alpha Ki of 1.09 pM. In contrast, complexed 125I-rHir was found to be a pure, non-competitive inhibitor having a Ki of 15.6 pM showing a ten-fold increase. These results demonstrate that covalently bound 125I-rHir still maintains potent alpha-thrombin affinity while losing minimal inhibitory capacity. Thus, successful modification of 125I-rHir serves as the foundation for future alternative applications for this potent inhibitor.

Synthesis of a novel small diameter polyurethane vascular graft with reactive binding sites.

Development of a small diameter (4 mm inner diameter [ID]) prosthetic vascular graft with functional groups accessible for covalent binding of recombinant hirudin (a potent anticoagulant) should create a more hemocompatible surface. The purpose of this study was to develop a technique for generating carboxylic acid groups on the surface of precast 4 mm ID poly-(carbonate urea)-urethane vascular grafts and to evaluate the accessibility of these groups. A polycarbonate based urethane with the chain extender 2,2-bis(hydroxymethyl)propionic acid was synthesized. A precast 4 mm ID poly(carbonate urea)-urethane vascular graft (Chronoflex [CF]; CardioTech International, Woburn, MA) was then placed into a 4% carboxylated polyurethane (cPU) solution (in 1% dimethyl acetamide) and incubated for 30 minutes (cPU graft). To determine the accessibility of the carboxylic acid groups, a standard textile technique using methylene blue dye was used. Macroscopic cross-sections, which were cut and evaluated for dye penetration, showed greatest concentration of carboxylic acid groups at the luminal and capsule surfaces, with minimal penetration into the mid-portion of the graft. Analysis of dye baths for absorbance reduction resulted in the cPU grafts having 3.7-fold and 5.4-fold more accessible carboxylic acid groups compared with untreated and dimethyl acetamide dipped CF grafts. Thus, a novel small diameter vascular graft has been developed that contains reactive carboxylic acid groups accessible for protein binding.

Bioengineering of a novel small diameter polyurethane vascular graft with covalently bound recombinant hirudin.

Development of a small diameter prosthetic vascular graft with surface based antithrombin properties should aid in maintaining early graft patency in small vessel reconstruction. The purpose of this study was to bind covalently a basecoat protein (canine serum albumin [CSAJ) and a potent antithrombin agent (recombinant hirudin [rHir]) to 4 mm inner diameter poly(carbonate urea) urethane grafts with reactive carboxylic acid groups (cPU). 125I-CSA was covalently bound to 1 cm length segments of cPU grafts using the carbodimide cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To bind 125I-rHir covalently, CSA was modified with the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) before linkage to the cPU surface with EDC (cPU-CSA-SMCC). 125I-rHir was modified with Traut's reagent and reacted with the cPU-CSA-SMCC surface, covalently linking 125I-rHir to surface bound CSA. 125I-CSA binding to the cPU graft surface (34,235 ng/segment) was ninefold, sevenfold, and 10-fold greater than controls with nonspecifically bound 125I-CSA. Covalent linkage of 125I-rHir to the cPU-CSA-SMCC surface (9,974 ng/segment) was 172, 192, and 142-fold greater than controls with nonspecifically bound 125I-rHir. Surface antithrombin properties were characterized using a chromogenic assay to measure residual thrombin activity. Evaluation of surface antithrombin activity showed significantly greater 131I-thrombin inhibition and binding by the cPU surface with covalently bound 125I-rHir, as compared with controls. Release of 125I-rHir from the cPU surface was minimal as compared with controls. Therefore, rHir can be covalently linked to a novel small diameter polyurethane vascular graft surface while maintaining its potent antithrombin properties.

Development of infection resistant polyurethane biomaterials using textile dyeing technology.

Infection is a major complication when using biomaterials such as polyurethane in the clinical setting. The purpose of this study was to develop a novel infection resistant polyurethane biomaterial using textile dyeing technology. This procedure results in incorporation of the antibiotic into the polymer, resulting in a slow, sustained release of antibiotic from the material over time, without the use of exogenous binder agents. Polycarbonate based urethanes were synthesized that contained either a non-ionic (bdPU) or anionic (cPU) chain extender within the polymer backbone and cast into films. The fluoroquinolone antibiotic ciprofloxacin (Cipro) was applied to bdPU and cPU using textile dyeing technology, with Cipro uptake determined by absorbance reduction of the "dyebath." These dyed bdPU/cPU samples were then evaluated for prolonged Cipro release and antimicrobial activity by means of spectrophotometric and zone of inhibition assays, respectively. Cipro release and antimicrobial activity by dyed cPU segments that were aggressively washed persisted over 9 days, compared with dyed bdPU and dipped cPU control segments that lasted < 24 hours. Dyed cPU segments, which remained in a static wash solution, maintained antimicrobial activity for 11 days (length of study), whereas controls again lost antimicrobial activity within 24 hours. Thus, application of Cipro to the cPU polymer by means of dyeing technology results in a slow sustained release of antibiotic with persistent bacteriocidal properties over extended periods of time.

Chemical and physical characterization of a novel poly(carbonate urea) urethane surface with protein crosslinker sites.

A major complication which occurs with implantable polyurethane biomaterials is bioincompatibility between blood and the biomaterial surface. Development of a novel biodurable polyurethane surface to which biological agents, such as growth factors or anticoagulants could be covalently bound, would be beneficial. The purpose of this study was to synthesize a novel poly(carbonate urea) urethane polymer with carboxylic acid groups which would serve as "anchor" sites for protein attachment. Physical characteristics such as tensile strength, initial modulus, ultimate elongation, tear strength, water/alcohol uptake and water vapor permeation were then evaluated and compared to other biomedical-grade polyurethanes. Covalent linkage of the blood protein albumin to this novel surface was then examined. A biodurable polycarbonate-based polyurethane containing carboxylic acid groups (cPU) was synthesized using a two step procedure incorporating the chain extender 2,2-bis(hydroxymethyl)-propionic acid (DHMPA). Tensile strength of this cPU film was 2.7 and 2.6 fold greater than both a polycarbonate-based polyurethane synthesized with a 1,4-butanediol chain extender (bdPU) and Mitrathane (Mit) controls, respectively. The cPU polymer also possessed 7.8 and 31 fold greater structural rigidity upon evaluation of initial modulus as compared to the bdPU and Mit, respectively. Ultimate elongation for the bdPU films was slightly higher than the cPU and Mit films, which had comparable elongation properties. The force required to tear the bdPU film was 1.9 and 32 fold greater than the cPU and Mit films, respectively. Alcohol solution uptake by all of the polyurethane segments increased with increasing alcohol concentrations, with the cPU having the greatest uptake. Water uptake was minimal for all the polyurethanes examined and was not affected by altering pH. Water vapor permeation was lowest for the cPU films as compared to both bdPU and Mit. Swelling the cPU in 50% ethanol prior to evaluation slightly increased water vapor permeation through the films. Covalent linkage of the radiolabelled blood protein albumin (125I-BSA) to the cPU segments incubated with the heterobifunctional crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was greatest in the higher percent of ethanol as compared to controls. These results serve as foundation for developing a novel poly(carbonate urea) urethane with physical characteristics comparable to other medical-grade polyurethanes while having protein binding capabilities.

Evaluation of a novel hirudin-coated polyester graft to physiologic flow conditions: hirudin bioavailability and thrombin uptake.

PURPOSE: Our laboratory has developed methods required to covalently bind recombinant hirudin (rHir) to the surface of polyester vascular grafts. Using alkaline hydrolysis of the polyester surface, carboxyl-binding sites are created on the outer periphery of each fiber. A series of static, in vitro experiments have demonstrated that surface-bound rHir rapidly removes and inhibits activated human alpha-thrombin from the reaction system; however, the performance of this modified graft material under physiologic flow conditions was undefined. METHODS: An in vitro flow loop was used to evaluate structural stability of the 125I-rHir and 131I-albumin covalently bound to the surface of 6 mm interior diameter crimped polyester grafts exposed to either constant flow (n = 4; shear rate, 300 sec(-1)) or pulsatile flow (n = 4; maximum shear rate, 780 sec(-1)) conditions for a 7-day period. In a separate series of experiments, the kinetics of thrombin-rHir interaction were evaluated through perfusion of 125I-rHir-coated grafts (n = 6) with 131I-thrombin for a 27-hour period under constant flow conditions. Identically prepared 125I-albumin-coated grafts (n = 3) were used as controls. RESULTS: Results of the stability experiments were independent of flow conditions, demonstrating moderate loss of both proteins, with rHir and albumin losses of 52.1% and 19.9% under constant flow and 49.1% and 21.6% under pulsatile flow, respectively. With results comparable with those of previous static experiments, rHir-coated grafts were significantly more effective at removing thrombin from the perfusion stream with 131I-thrombin binding densities of 3.08 +/- 0.61 and 0.64 +/- 0.04 NIHU/cm2 (p < 0.01) for rHir-coated and albumin-coated grafts, respectively. Estimates of the total amount of thrombin inactivated during the perfusion period similarly demonstrated a marked difference between the rHir-coated and control graft segments (125 +/- 8 vs. 3 +/- 14 NIHU; p < 0.005). CONCLUSIONS: These in vitro flow results illustrate that polyester grafts with covalently bound rHir can provide significant reductions in local thrombin concentration under physiologic flow conditions, and can serve as a foundation with which to understand the performance of these grafts when implanted in vivo under physiologic flow and shear rates.

Modification of polyethylene terephthalate (Dacron) via denier reduction: effects on material tensile strength, weight, and protein binding capabilities.

Thrombosis remains a significant and potentially catastrophic complication of polyethylene terephthalate (Dacron) prosthetic vascular graft implantation. Numerous attempts have been made to create a novel surface that reduces the adverse effects of blood interaction with the material. The purpose of this study was to create reactive groups on Dacron without significantly altering the chemical and physical properties of the biomaterial. These groups would then serve as "anchor sites" for covalent attachment of the blood protein albumin to the surface, thus creating a more biocompatible surface. Denier reduction, an established textile chemistry procedure that creates carboxyl groups on the fiber surface via hydrolysis of the material, was performed at 100 degrees C using sodium hydroxide concentrations of 0.5, 1.0, 2.5, and 5.0% (treated materials referred to as 0.5% hydrolyzed etc.). Tensile strength determination of hydrolyzed materials revealed no statistically significant difference in material strength between control, 0.5, and 1.0% hydrolyzed materials; the 2.5 and 5.0% hydrolyzed materials had significant strength loss as compared to the controls. Significant fiber weight loss occurred in the 1.0, 2.5, and 5.0% hydrolyzed Dacron segments. The 0.5% hydrolyzed material did not have any significant weight loss. Covalent linkage of 125I-albumin to these modified materials using the crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride (EDC) resulted in the 0.5% hydrolyzed material having the greatest protein binding (330 ng/mg Dacron, 2,4-fold greater than control). Incubation of the 0.5% hydrolyzed material with EDC and various concentrations of 125I-albumin resulted in the 14.80 microM solution permitting the greatest binding per milligram Dacron (330 ng/mg Dacron). Scanning electron microscopy, performed blindly, revealed no change in the 0.5% hydrolyzed Dacron as compared to untreated Dacron. The 5.0% hydrolyzed Dacron, however, had noticeable structural damage on the outer periphery of the fiber surface. Observation of the untreated Dacron with nonspecifically bound albumin showed scattered areas of albumin adherent to the fiber surface whereas covalent linkage of albumin to the 0.5% hydrolyzed Dacron via EDC crosslinking showed numerous albumin moieties on each fiber. This study demonstrates that a clinically accepted biomaterial (Dacron) can be chemically modified, without significantly altering the physical and chemical characteristics of the biomaterial, in order to covalently bind albumin to the fiber surface. Thus, these results serve as foundation for creating potential novel biomaterials without significantly altering the properties of the original biomaterial.

Identification of multiple genes with altered expression at the distal anastomosis of healing polytetrafluoroethylene grafts.

PURPOSE: Anastomotic intimal hyperplasia remains a significant cause of delayed prosthetic arterial graft failure. Prior studies have identified several genes with altered expression within the hyperplastic region at the downstream polytetrafluoroethylene arterial anastomosis as compared with normal arteries. The purpose of the current study was to determine the sequence of early gene-related events at the distal anastomosis of an in vivo prosthetic arterial graft model. Messenger RNA (mRNA) differential display was used to screen for alterations in gene expression between anastomotic sites and control arterial segments. METHODS: Six carotid interposition 6-mm expanded polytetrafluoroethylene grafts were placed in mongrel dogs, with the intervening carotid artery segment serving as the baseline control. Five days after graft implantation, the distal anastomotic artery segments were harvested and total RNA was isolated from both the intervening normal arteries and anastomotic segments. Differential mRNA display was used to identify candidate complementary DNA (cDNA) clones with expression that differed in anastomotic segments as compared with normal intervening arteries. Northern blot analysis confirmed alteration of gene expression. The cDNA clones were sequenced, and gene databases were searched. Novel sequences were used as probes for screening human cDNA libraries. RESULTS: Approximately 7000 mRNA species were screened, and 26 candidate clones were obtained. Northern blot analysis showed altered gene expression in 10 (38%) of the clones, undetectable signals in 13 (50%), and nonregulation in 3 (12%). Seven clones with 92% homology at the nucleotide level to human alpha1 (III) procollagen gene and novel sequence were expressed only at the distal anastomosis. A clone representing apolipoprotein J and a novel sequence had increased expression at the distal anastomosis of 364% +/- 236% and 156% +/- 47%, respectively (mean percentage, control +/- standard deviation). CONCLUSIONS: These studies identified genes with expressions that increased or were exclusive to the distal anastomosis of healing prosthetic arterial grafts in an in vivo prosthetic arterial graft model. Type III collagen may contribute significantly to the composition of the extracellular matrix associated with intimal hyperplasia by increasing lesion volume. Apolipoprotein J, through its association with proteases, may modulate some of the matrix changes seen early after grafting.

Covalent linkage of recombinant hirudin to a novel ionic poly(carbonate) urethane polymer with protein binding sites: determination of surface antithrombin activity.

Surface thrombus formation on implantable biomaterials such as polyurethane is a major concern when utilizing these materials in the clinical setting. Thrombin, which is responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous surface modification strategies in an effort to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) onto a novel polyurethane polymer synthesized with carboxylic acid groups which served as protein attachment sites. The in vitro efficacy of thrombin inhibition by this novel biomaterial surface was then evaluated. Bovine serum albumin (BSA), which was selected as the basecoat protein, was reacted with sulfo-SMCC in a 1:50 molar ratio. This BSA-SMCC complex was then covalently linked to the carboxylated polyurethane (cPU) surface via the crosslinker EDU (cPU-BSA-SMCC). This cPU-BSA-SMCC surface was then reacted with Traut's-modified 125I-rHir, a procedure which created free sulfhydryl groups on rHir (cPU-BSA-SMCC-S-125I-rHir). Using these crosslinking procedures, the cPU-BSA-SMCC-S-125I-rHir segments bound 188 +/- 40 ng/cm2 (n = 60) whereas the controls with non-specifically bound 125I-rHir (Mitrathane + EDC + BSA + 125I-rHir-SH and cPU-BSA + 125I-rHir-SH) bound 13 +/- 8 ng/cm2 and 4 +/- 8 ng/cm2, respectively. Evaluation of these cPU-BSA-SMCC-S-125I-rHir segments for 131I-thrombin inhibition using a chromogenic assay for thrombin showed that a maximum of 2.64 NIHU thrombin was inhibited in contrast to the controls which inhibited bound 0.76 and 0.70 NIHU. Controls with nonspecifically bound 125I-rHir also had 0.31 and 0.76 NIHU 131I-thrombin adherent to their respective surfaces whereas the maximum 131I-thrombin binding to the cPU-BSA-SMCC-S-rHir segments was 1.51 NIHU. Exposure to 131I-thrombin did not result in any release of covalently bound 125I-rHir from the cPU-BSA-SMCC-S-125I-rHir segments. Thus, these results demonstrate that rHir can be covalently bound to this novel polyurethane surface and still maintain potent antithrombin activity.

Glycoconjugate mediated endothelial cell adhesion to Dacron polyester film.

PURPOSE: The purpose of this study was to explore new strategies for enhancing specific cell type attachment to biomaterials using immobilized lectins for cell surface glycoconjugates. The lectin Ulex europaeus I (UEA I) has a high affinity for human vascular endothelial cell surface glycoconjugates. METHODS: UEA I was covalently bound to polyethylene terephthalate (Dacron) with the cross-linking agent 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride to achieve oligosaccharide-mediated endothelial cell attachment to this otherwise nonadherent surface. RESULTS: Experiments with radiolabeled UEA I demonstrated covalent linkage of as much as 1.35 micrograms/cm2. The lectin binding site is available after the reaction, as demonstrated in experiments a neoglycoprotein. Adhesion studies reveal a 100-fold increase in endothelial cell attachment for the UEA I/polyethylene terephthalate surface (99.7 +/- 29.6 cells/high-power field) when compared with untreated (0.7 +/- 0.5), crosslinking agent (0.4 +/- 0.3), and denatured UEA I (1.2 +/- 1.1) control groups. Five vascular endothelial cell lines adhered to the UEA I/polyethylene terephthalate surface, whereas monocytes, smooth muscle cells, and fibroblasts did not. CONCLUSION: These results imply new strategies for endothelialization of prosthetic grafts and promotion of selective cell adherence to biomaterials, with emphasis on carbohydrate interactions. Moreover, this experimental system offers a model for exploring the biologic significance of the endothelial cell-UEA I ligand.

In vivo testing of an infection-resistant vascular graft material.

Present prosthetic arterial conduits continue to suffer the clinically and economically catastrophic complication of infection. We recently described a novel technique for binding quinolone antibiotics to Dacron based on principles of textile chemistry. This thermofixation procedure ("pad/heat") utilizes the limited fibrophilic characteristics of the quinolone antibiotic ciprofloxacin (Cipro) to permit pad/heat application and allowed controlled, sustained release from Dacron in several in vitro assays. The objective of this study was to test this infection-resistant prosthetic vascular graft material in an in vivo model. Dacron segments (1 cm2, either plain, dipped into antibiotic immediately prior to implantation, or Cipro pad/heat treated) were implanted in the dorsal subcutaneous tissue of the rabbit and directly contaminated with 10(6) Staphylococcus aureus. After 1 week, the samples were sterily harvested. Wounds were blindly graded on a scale from 1 (no evidence of infection, good tissue incorporation) to 4 (suppurative infection extending outside of the graft pocket, no gross tissue incorporation). Plain Dacron was easily infected in this model (mean grade 3.1 +/- 0.6, 92% culture positive). Notably, however, a significant (P < 0.05) wound grade difference between the dipped (2.3 +/- 1.0) and pad/heat (1.4 +/- 0.6) samples was demonstrated. Determination of adherent bacteria present on the implanted Dacron pieces by sonication and culture studies again revealed a significant difference between the dipped (56% culture positive) and pad/heat (12% culture) groups (P < 0.025). Histologic studies confirmed good tissue incorporation of the pad/heat samples. This project opens new avenues in the development of infection-resistant biomaterials.

Application of the quinolone antibiotic ciprofloxacin to Dacron utilizing textile dyeing technology.

Prosthetic arterial graft infection continues to be a significant and often devastating complication of vascular surgery. The organisms Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) are the primary pathogens causing acute and late graft infections, respectively. The objective of this study was to develop an infection-resistant prosthetic arterial graft by applying the bacteriocidal quinolone antibiotic ciprofloxacin to polyethylene terepthalate (Dacron) via thermofixation (pad/heat), a new application method founded on established textile procedures. We hypothesize that the limited fibrophilic characteristics of ciprofloxacin will permit binding to Dacron and at the same time allow persistent controlled release over an extended period of time. Using pad/heat technology, 33 micrograms (+/- 2.97 micrograms, n = 12) of ciprofloxacin was successfully bound to a 1-cm2 piece of woven Dacron. A full complement of microbiologic assays demonstrated superior, sustained antistaphylococcal activity of the pad/heat Dacron when compared to Dacron dipped into an equivalent concentration of ciprofloxacin. The sustained antimicrobial efficacy of ciprofloxacin pad/heat-treated Dacron opens new avenues in the development of infection-resistant biomaterials based on an understanding of textile chemistry.

Isolation of genes differentially expressed at the downstream anastomosis of prosthetic arterial grafts with use of mRNA differential display.

PURPOSE: Downstream anastomotic intimal hyperplasia in prosthetic arterial grafts remains a major cause of delayed graft failure. The new method of messenger RNA (mRNA) differential display was used to screen numerous genes to gain insight into the molecular mechanisms of intimal hyperplasia. METHODS: Fifty-centimeter-long 8 mm expanded polytetrafluoroethylene grafts were placed in four mongrel dogs from the carotid artery to the distal abdominal aorta. At 3 months the distal anastomoses and adjacent normal aortas were harvested; a portion was taken for histologic examination, and total RNA was isolated from the remainder. Differential mRNA display was used to identify candidate cDNA clones whose expression differed in anastomotic intimal hyperplasia as compared with adjacent unaffected aorta. The clones were sequenced, and national gene databases were searched. Northern blot analysis confirmed alteration of gene expression. RESULTS: Approximately 5000 mRNA species were screened, and 11 candidate clones were obtained. DNA sequence revealed homology of five clones to known gene sequences. Homologous genes included an interferon-gamma-induced human gene, (IGUP I-5111), alpha-1 protease inhibitor gene, human retinoblastoma susceptibility gene, and human creatine kinase gene (two clones). Northern blot analysis revealed altered gene expression in 4 of 11, nonregulation in 1 of 11, and undetectable signals in 6 of 11. Expression of the clone representing IGUP I-5111 in the segment of intimal hyperplasia was found to be decreased over threefold to only 31% +/- 16.4% SE of the level seen in normal aorta. CONCLUSIONS: The technique of mRNA differential display has identified differences in gene expression in an in vivo model of anastomotic intimal hyperplasia. Expression of RNA with homology to an interferon-gamma-induced human gene was consistently decreased within the hyperplastic region at the downstream polytetrafluoroethylene arterial anastomosis.