Effect of heparin-PVA hydrogel on platelets in a chronic canine arterio-venous shunt

Polyvinyl alcohol (PVA) hydrogel, with or without heparin, was reactive towards canine platelets in a chronic arteriovenous shunt as demonstrated by an increase in platelet regeneration time, a systemic decrease in platelet count and transient decrease in platelet serotonin content. Immobilized heparin (heparin-PVA) had no effect whereas unmodified polyethylene was found to be unreactive despite similar levels of platelet deposition as measured by SEM and a higher in vitro reactivity (J. Biomed. Mater. Res., this issue). Twenty-centimeter lengths of hydrogel coated polyethylene tubing were inserted between the arterial and venous portions of the shunt and left in place for 4-6 days, without the complicating artifacts of anticoagulation, anesthesia, or surgical intervention. Regeneration time was measured as the return to normal platelet cyclooxygenase (co) activity after a single 240-mg dose of aspirin, with co activity measured in vitro as malondialdehyde production. Although measuring new platelet production, regeneration time is an indirect measure of platelet consumption, so that the reduced regeneration time seen here was presumed to reflect enhanced material associated consumption and thromboembolism. Like other hydrogels, PVA does not appear to be "thromboadherent" but it does appear thrombogenic. Immobilized heparin had no additional effect, presumably because the platelet response was dominated by the reactivity of the underlying substrate.     

Heparin-poly(ethylene glycol)-poly(vinyl alcohol) hydrogel: preparation and assessment of thrombogenicity.

Heparin was immobilized on to poly(vinyl alcohol) hydrogel (PVA) through the free isocyanate end-group on a poly(ethylene glycol) (PEG2000) which had been previously covalently linked to the hydrogel via a urethane moiety. The intention was to reduce the platelet reactivity of the PVA while also suppressing fibrin formation. Elemental nitrogen analysis revealed that the total amount of bound heparin was 19 +/- 7 mumol/g of dried gel. An increase in the in vitro whole blood clotting time of PVA was observed. This was attributed to bound heparin, as the elution rate of heparin from the gel (23 pmol/m2 min) was too low to produce a significant bulk concentration to interfere with fibrin formation. Ex vivo assessment using a chronic canine A-V shunt showed that the bound heparin hydrogel had no effect on the drop in the number of platelets induced by PVA hydrogel, but increased the fractional rate of platelet destruction from approximately 0.35/d to an average value of 0.42/d.     

Characterization of transient platelet contacts on a polyvinyl alcohol hydrogel by video microscopy.

Acridine orange labelled, washed human platelets were counted and tracked on polyvinyl alcohol (PVA), heparin-PVA and polyethylene (PE)-coated coverslips with a view to understand why transient contact on the PVA hydrogels lead to elevated platelet activation and consumption relative to polyethylene. Over the 4 min of initial contact that was studied, platelet adhesion was higher on PE than on PVA or heparin-PVA at both 40 and 200 s(-1), as expected, regardless of whether the surfaces were pre-treated with albumin or fibrinogen. Not all platelets appearing to make contact with the surface, actually attached. For example, less than 2% of the platelets contacting albumin pre-treated PVA (at 40 s(-1)) remained adherent at the end of the initial 60 s observation time, while the corresponding number for PE was greater than 9%. A greater fraction of the platelets remained adherent at the higher shear rate or with fibrinogen pre-treatment, but the difference between PVA and PE remained similar: for example, with fibrinogen pre-treatment at 200 s(-1), approximately 25% of the platelet contacts resulted in adhesion on PVA while 66% did so on PE. While net platelet adhesion was less for the hydrogels, than for PE, the total number of contacts (adherents + non-adherents) were more comparable and unexpectedly higher for albumin pre-treatment than for fibrinogen. Net platelet adhesion is but one component of the total platelet interaction with a material surface. Fluorescent video microscopy has been shown to be a useful, albeit not unequivocal, method for assessing the platelets that make contact with but do not adhere to a surface. reserved     

Platelet consumption by polyvinyl alcohol coated tubing in canines

Polyvinyl alcohol (PVA)-coated polyethylene tubing, with or without immobilized heparin, caused severe thrombocytopenia and enhanced the production of new platelets when inserted in a chronic arteriovenous shunt in canines. A similar length of uncoated polyethylene tubing neither lead to thrombocytopenia nor significantly enhanced platelet regeneration, relative to the shunt only without a test section. Platelet regeneration was monitored by the malondialdehyde assay, which was assumed to make a distinction between "new" and "old" platelets. This distinction was combined with the platelet count values to enable calculation of the cumulative consumption curve and the initial fractional consumption rate in the presence of a non constant platelet count. The resulting initial fractional consumption rates were: 34%/day for PVA, 20.5%/day for polyethylene, and 18%/day for the shunt only blank.     

Thrombin and albumin adsorption to PVA and heparin-PVA hydrogels. I. Single protein isotherms.

More radiolabeled thrombin was adsorbed to heparin-polyvinyl alcohol (PVA) than to PVA, consistent with a specific interaction with the immobilized heparin. The maximum surface concentration on heparin-PVA was estimated to be approximately 450 nmol/m2 with an apparent affinity constant (Ka) of 2.5 microM-1; on PVA, the plateau concentration was 10 nmol/m2 with a Ka less than 1 nM-1. There was little difference in bovine serum albumin (BSA) adsorption between PVA and heparin-PVA. Interestingly, thrombin adsorption to polyethylene was indistinguishable from that to PVA despite the large difference in surface chemistry. BSA adsorbed to polyethylene with higher affinity than to the hydrogels, although the plateau concentrations were comparable. The adsorbed thrombin was biologically inactive at least towards chromogenic substrate, with the residual activity on PVA unaffected by subsequent incubations with antithrombin III. PVA and heparin-PVA presented a heterogeneous and complex substrate for interaction with proteins. The adsorbed protein was likely present in multiple states depending on the groups with which it interacted.     

Inactivation of thrombin in heparin-PVA coated tubes

Heparin, immobilized to polyvinyl alcohol by reaction with glutaraldehyde (heparin-PVA), retained its ability to accelerate the antithrombin III inactivation of thrombin, in a recirculating flow loop using heparin-PVA coated polyethylene tubes. The extent of inactivation, for a constant flow time, was approximately constant over ten cycles of exposure to thrombin and antithrombin III, suggesting that the immobilized heparin was reusable, as expected from the catalytic nature of non-immobilized heparin. Assessment of the chromogenic substrate activity of adsorbed thrombin and the extent of displacement were less conclusive with the implication that thrombin is adsorbed to heparin-PVA or PVA without heparin in multiple states.     

Patency of heparin-PVA coated tubes at low flow rates

The patency of heparin-PVA coated 1.14 mm i.d. polyethylene tubes was greater than that of control tubes without heparin in a novel test section that enabled ex vivo evaluation in dogs at low flow and shear rates (2-10 ml/min). Low shear rates were achieved by diverting a small portion of a chronic A-V shunt flow through Y-connectors into the small diameter test tubing. For shear rates in the range of 200-650 s-1 half the heparin-PVA tubes remained patent for longer than 72 min whereas half the control tubes (PVA only) only remained patent for longer than 30 min. At higher shear rates (500-1000 s-1), the 50% patency times for heparin-PVA and PVA were 225 and 123 min respectively. The increased patency at the higher shear rates was attributed more to the effect of the connector than just shear rate. The higher shear rates were achieved by changing from a Y-connector in which the small diameter side-branch was cut flush to the larger diameter main line to one in which the small diameter test tubing protruded into the lumen of the main line; the latter 'protruding' connector was presumed to result in less platelet pre-activation than the other. This method has been found useful for assessing the thrombogenicity of heparinized tubes at low flow rates.     

Surface modification with PEO-containing triblock copolymer for improved biocompatibility: in vitro and ex vivo studies

Poly(ethylene oxide) (PEO) has been frequently used to modify biomaterial surfaces for improved biocompatibility. We have used PEO-polybutadiene-PEO triblock copolymer to graft PEO to biomaterials by gamma-irradiation for a total radiation dose of 1 Mrad. The molecular weight of PEO in the block copolymer was 5000. In vitro study showed that fibrinogen adsorption to Silastic, polyethylene, and glass was reduced by 70 to approximately 95% by PEO grafting. On the other hand, the reduction of fibrinogen adsorption was only 30% on expanded polytetrafluoroethylene (e-PTFE). In vitro platelet adhesion study showed that almost no platelets could adhere to PEO-coated Silastic, polyethylene, and glass, while numerous platelet aggregates were found on the ePTFE. The platelet adhesion in vitro corresponded to the fibrinogen adsorption. When the PEO-grafted surfaces were tested ex vivo using a series shunt in a canine model, the effect of the grafted PEO was not noticeable. Platelet deposition on ePTFE was reduced by PEO grafting from 8170 +/- 1030 to 5100 +/- 460 platelets 10(-3) microm2, but numerous thrombi were still present on the PEO-grafted surface. The numbers of platelets cumulated on Silastic, polyethylene, and glass were 100 +/- 80, 169 +/- 35, and 24 +/- 22 platelets 10(-3) microm2, respectively. This is about 35% reduction in platelet deposition by PEO grafting. While the numbers of deposited platelets were small, the decreases were not as large as those expected from the in vitro study. This may be due to a number of reasons which have to be clarified in future studies, but it appears that in vitro platelet adhesion and fibrinogen adsorption studies may not be a valuable predictor for the in vivo or ex vivo behavior of the PEO-grafted surfaces.     

Permeability of a heparin-polyvinyl alcohol hydrogel to thrombin and antithrombin III

The diffusivities of thrombin and antithrombin III in a heparin-polyvinyl alcohol hydrogel were estimated and used to demonstrate that diffusion limits the effectiveness of the immobilized heparin in the interior of such hydrogels. Diffusivities were calculated from permeabilities and partition coefficients measured with films in a diffusion chamber apparatus. The diffusion coefficients were estimated to be 6 +/- 4 X 10(-8) cm2/s for thrombin and 4 +/- 2 X 10(-8) cm2/s for antithrombin III in 10% gel membranes with or without immobilized heparin. Using the diffusivity of thrombin and a Thiele-type modulus, the effectiveness factor of a spherical heparin-PVA bead used to accelerate the inactivation of thrombin by antithrombin III was found to be 4-9% (diameter range 250-150 micron). While indicating that diffusion of thrombin limited the full utilization of the immobilized heparin, these values for the effectiveness factor could not completely account for the low apparent heparin activity (0.2%) in a thrombin time test of heparin-PVA "beads" (J. Biomed. Mater. Res., 17, 359 (1983]. Other factors such as the immobilization chemistry or the diffusion of thrombin-antithrombin III complex must be considered for a full explanation of the thrombin time results.     

Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge

The blood compatibility of poly(ethylene oxide) (PEO)-grafted and heparin (Hep) immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on heparin-immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on heparin-immobilized PUs, and the amount of interleukin-6 (IL-6) released from PBMCs stimulated with surface-modified PUs decreased with a decrease in PBMC adhesion.     

Synthesis and antithrombogenicity of heparinized polyurethanes with intervening spacer chains of various kinds

Heparin was immobilized to polyetherurethaneurea membrane by covalent or ionic bondings with intervening spacer chains having different lengths and different terminal functional groups. The amount of immobilization of heparin and the release rate of immobilized heparin were controlled by the nature and the mode of bonding of spacer chains. The heparinized polyetherurethaneurea membranes became more in vitro antithrombogenic and suppressed more strongly the adhesion and activation of platelets, as the amount of immobilization increased. It was also shown that the membrane to which the low-molecular-weight fraction of heparin was immobilized was less stimulating to platelets.     

In vitro platelet adhesion and in vivo antithrombogenicity of heparinized polyetherurethaneureas

We investigated in-vitro platelet adhesion to polyetherurethaneureas, to which heparin was bound covalently or by ionic bonding. When heparin was bound to polymers, platelet adhesion and platelet activation upon adhesion were suppressed with the increasing content of bound heparin in the polymer. Platelets were activated upon adsorption to different degrees according to the method of heparinization. The platelet adhesion and the platelet activation upon adhesion appeared to be regulated by the electrostatic repulsion between platelet and anionic surface of covalently or ionically heparinized polymer, rather than by the physiological action of bound heparin. The effect of the method of heparinization seemed to be related to the molecular heterogeneity of heparin. Heparinized polyurethanes which interacted very weakly with platelets in vitro were tested for in-vivo antithrombogenicity. The test was carried out by the implantation of a suture of the heparinized polyurethane into canine veins. Ionically heparinized polyurethane did not form a thrombus and maintained a smooth surface over a long period. On the other hand, covalently heparinized polyurethane formed a small amount of thrombus and grew endothelial cells from the insertion point.     

Preparation and thrombogenicity of alkylated polyvinyl alcohol coated tubing.

A polyvinyl alcohol hydrogel (PVA) was reacted with a C18 isocyanate at 80 degrees C in dimethyl formamide (DMF) in order to improve the platelet reactivity of the hydrogel through an influence on albumin adsorption or retention. A C4 isocyanate was used as a control. Surface coverage by XPS appeared to be approximately 100% for both C4 and C18 modified surfaces, although the limited solubility of C18 isocyanate in DMF may have resulted in a nonuniform surface. Relative to PVA or the solvent treated control, octadecylation resulted in increased albumin adsorption (from a single protein solution) and increased retention when the adsorbed albumin was exposed to a fibrinogen solution. However, octadecylation did not obviate the platelet reactivity problem in preliminary studies: systemic platelet counts were reduced by about half over 4 days in a canine AV shunt experiment and the initial rate of platelet destruction for C18-PVA was greater (36%/day) even than for the solvent-treated PVA. Surprisingly in preliminary studies butylation of PVA resulted in little or no thrombocytopenia and did not appear to increase significantly the fractional rate of platelet destruction relative to the shunt only blank. It is presumed that the nonspecific effect of alkylation (independent of chain length) was the dominant contribution to the reduced platelet reactivity. A similar effect of C18-PVA presumably would have been observed had the limited solubility of C18 isocyanate not precluded a uniform surface coverage.     

Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge

The blood compatibility of poly(ethylene oxide) (PEO)-grafted and heparin (Hep) immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on heparin-immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on heparin-immobilized PUs, and the amount of interleukin-6 (IL-6) released from PBMCs stimulated with surface-modified PUs decreased with a decrease in PBMC adhesion.     

Antithrombogenic surfaces: characterization and bioactivity of surface immobilized PGE1-heparin conjugate

A covalently bonded conjugate of commercial grade heparin and prostaglandin E1 (PGE1) was synthesized to prevent both fibrin formation and platelet aggregation during thrombus formation. The PGE1-heparin conjugate was immobilized on an imidazole carbamate derivatized sepharose bead surface through hydrophilic spacer groups (diamino-terminated polyethylene oxides). One end of the spacer group was coupled to the derivatized surface through a urethane bond between the amine group of the spacer and the derivatized surface. The free amine group of the immobilized spacers was coupled to a carboxylic group of the PGE1-heparin conjugate through an amide bond. Bioactivity of the immobilized conjugate (heparin activity) was measured in terms of increased clotting times (thrombin time assay) and for the inactivation of Factor Xa. Bioactivity of the immobilized compound (PGE1 activity) was analyzed by platelet adhesion and platelet release reactions using C14-5-hydroxytryptamine (5-HT). The conjugate immobilized via the C2 spacer showed the highest incidence of platelet adhesion, 5-HT released and the lowest activity for coagulation factors. In contrast, the 1000 and 4000 immobilized systems showed a significant reduction in platelet activation, while having the greatest effect on coagulation factors. The results of these experiments imply that the immobilized conjugate is active in preventing both pathways of thrombus formation, and the efficacy is improved through the use of long-chain hydrophilic spacer groups.     

Controlled release of heparin from polypyrrole-poly(vinyl alcohol) assembly by electrical stimulation

A surface modification technique was developed for the covalent immobilization of poly(vinyl alcohol) (PVA)-heparin hydrogel onto electrically conductive polypyrrole (PPY) film with the objective of achieving controlled release of heparin. First, aldehyde groups were introduced onto PPY film through poly(ethylene glycol) monomethacrylate graft copolymerization and subsequent oxidation in acetic anhydride and dimethyl sulfoxide mixture. Then, the prepared PVA-heparin hydrogel was cast onto the PPY film and covalently immobilized to the film through the reaction between the aldehyde groups on the PPY film and the hydroxyl groups of PVA. X-ray photoelectron spectroscopy was used to characterize the surface-modified film after each stage. The strong attachment of the PVA-heparin layer on the PPY film was confirmed by peel test and scanning electron microscopy. The release behavior of heparin from the substrate with and without electrical stimulation was studied and the experimental results showed that the heparin release rate from the prepared substrate using an electric current of 3.5 mA is twofold higher than that without current.     

Measurement of platelet loss in the blood compatibility assessment of biomaterials

In the assessment of the in vitro blood compatibility of biomaterials, platelet loss is often attributed solely to platelet adhesion and consideration is not given to platelets lost in platelet aggregate formation. In order to distinguish between those platelets lost to adhesion and those lost to aggregate formation, the Wu and Hoak method for the quantification of circulating platelet aggregates in patients has been modified to establish a new test procedure. This procedure, which measures both platelet adhesion (PA) in the absence of platelets lost to aggregate formation and also the tendency of a material to induce aggregate formation, has been used to evaluate the influence of a range of polyamides and a hydrogel. The evaluation demonstrated the ability of polymers to induce readily platelet aggregates during in vitro blood-material contact. The sensitivity of the aggregate measurement was exemplified by the polyamides, where PA was similar for materials of different porosity but platelet aggregate formation increased significantly with porosity. The importance of considering platelets lost to aggregate formation was emphasized with the hydrogel, where PA was low.     

Improved haemocompatibility of cysteine-modified polymers via endogenous nitric oxide.

A novel method for improving the haemocompatibility of biomedical materials through endogenous nitric oxide (NO) is presented. L-cysteine was covalently immobilized onto two biomedical polymers: polyurethane (PU) and polyethylene terephthalate (PET). The L-cysteine content on the polymers was approximately 5-8 nmol/cm2 as quantified via a chemiluminescence-based assay. The haemocompatibility of the modified polymers was evaluated in terms of the number of adhered platelets when exposed to a platelet suspension labeled with Cr51. Platelet adherence on the L-cysteine-modified polymers was reduced more than 50% as compared to the control (glycine-modified polymers) when the platelet suspension contained plasma constituents. No difference in platelet adhesion was observed in the absence of plasma constituents. Further experiments demonstrated that NO was easily transferred to the L-cysteine-modified polymers from S-nitroso-albumin in PBS buffer. The NO was then released from the polymer. NO transfer or release was not observed for the control. The results suggest that L-cysteine-modified polymers are effective in reducing platelet adhesion via the transfer of NO from endogenous S-nitrosoproteins in plasma to the polymer followed by the subsequent release of NO. Thus, exploiting endogenous NO is a viable option for improving the haemocompatibility of biomaterials.     

Glow discharge plasma treatment of polyethylene tubing with tetraglyme results in ultralow fibrinogen adsorption and greatly reduced platelet adhesion

Previous studies from our lab have shown that fibrinogen adsorption (Gamma(Fg)) must be reduced below 10 ng/cm(2) to significantly reduce platelet adhesion, and that radio frequency glow discharge (RFGD) treatment of polymeric films in the presence of tetraethylene glycol dimethyl ether (tetraglyme) can reduce Gamma(Fg) to the desired ultralow value. In this report, the effects of RFGD coatings of tetraglyme on the lumenal surface of PE tubing on Gamma(Fg) and on blood interactions both in vitro and ex vivo are described. Gamma(Fg) on the tetraglyme-coated PE tubing was reduced to the desired ultralow level (<10 ng/cm(2)), and we also observed a significant decrease in adsorption of von Willebrand's factor. In vitro platelet adhesion from washed platelet suspensions, platelet rich plasma, or whole blood to tetraglyme-coated PE tubing was decreased compared to PE, polyurethane, or silicone rubber tubes. In addition, thrombin generation by platelets adherent to tetraglyme-coated PE was also much less than by platelets adherent to PE. When inserted in an ex vivo carotid artery-carotid artery shunt in sheep, the RFGD tetraglyme-coated PE exhibited a very low number of adherent platelets compared to heparin-coated, chromic acid-etched, or plain PE. The RFGD tetraglyme-coated PE tubes exhibited high protein and platelet resistance in vitro, and high platelet resistance ex vivo. The improved hemocompatibility is attributed to the unique chemical structure of RFGD tetraglyme that makes it highly protein resistant.     

Platelet adhesion to heparin coated oxygenator fibers under in vitro static conditions: impact of temperature

Heparin coating of cardiopulmonary bypass (CPB) circuitry may attenuate the platelet consumption associated with CPB. We investigated the effect of temperature on the interaction between platelet and heparin coated surfaces under in vitro static conditions. Heparin coated and non coated oxygenator fibers were incubated with heparinized whole blood at 37 degrees C and 22 degrees C. The incubation time was set at 30, 60, 180, and 300 minutes. The number of platelets adhering to each fiber was assessed with enzyme immunoassay using monoclonal antibody against platelet receptor protein CD 61(GPIIbIIIa). As an index of platelet activation, plasma soluble(s) P-selectin levels were measured by enzyme-linked immunosorbent assay. Under normothermia, the number of adherent platelets on the non coated surface increased significantly after 300 min of incubation. Platelet adhesion was reduced significantly by heparin coating of the surface and was kept constant after 300 min. Under hypothermia, heparin coating was also associated with significant reduction of platelet adhesion. The levels of sP-selectin did not correlate with the extent of platelet adhesion. Our results suggest that heparin coating is effective in decreasing platelet adhesion to the synthetic surface tested regardless of the temperature under static conditions. Inhibition of platelet activation on the heparin coated surface may be masked by standard dose heparinization.