Membrane-mimetic films containing thrombomodulin and heparin inhibit tissue factor-induced thrombin generation in a flow model

Membrane-mimetic thin films containing thrombomodulin (TM) and/or heparin were produced and their capacity to inhibit thrombin generation evaluated in a continuous flow system. Tissue factor (TF) along with TM and heparin were immobilized in spatially restricted zones as components of a membrane-mimetic film. Specifically, TF was positioned as an upstream trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream portion of test films. Peak and steady-state levels of thrombin were decreased by antithrombin III (ATIII), as well as by surface bound heparin and TM. Although physiologic concentrations of ATIII have the capacity to significantly inhibit thrombin activity, surface bound TM and heparin nearly abolished steady-state thrombin responses. In particular, surface bound TM appears to be superior to heparin in reducing local thrombin concentrations. These studies are the first to demonstrate the additive effect of surface bound heparin and TM as a combined interactive strategy to limit TF-induced thrombin formation.     

Catalytic efficiency of a thrombomodulin-functionalized membrane-mimetic film in a flow model

The protein C anticoagulant pathway generates an "on demand" physiologic anticoagulant response, which is initiated when thrombin binds to thrombomodulin (TM), a transmembrane protein constitutively expressed by endothelial cells. A stable, protein C activating membrane-mimetic film was produced on a polyelectrolyte multilayer (PEM) by in situ photopolymerization of a phospholipid assembly containing TM. The monoacrylated phospholipid monomer was initially synthesized and prepared as unilamellar vesicles with varying molar concentrations of TM. Membrane-mimetic films were constructed on planar substrates with defined surface concentrations of catalytically active TM. 125I-labeled radiolabeling demonstrated little change in TM surface concentration over periods of up to 4 weeks. We utilized a parallel plate flow system to investigate the effects of simulated arterial (500 s(-1)) and venous (50 s(-1)) shear rates and TM surface concentration (0-1400 fmol cm(-2)) on the rate and extent of activation of protein C. The rate of production of activated protein C increased with shear rate and TM surface content. However, in agreement with an analysis of reaction kinetics and mass transfer, experimental results demonstrate that reaction rates become saturated at TM surface densities greater than or equal to 800 fmol cm(-2). We believe that the design of membrane-mimetic films that have the capacity to activate the protein C pathway will provide a useful strategy for generating "actively" antithrombogenic surfaces.     

Estimation of surface-bound heparin activity: a comparison of methods

We compared two assays for estimating the amount of active heparin bound to a catheter surface: 1) a kinetic assay based on the inactivation of thrombin by antithrombin III, and 2) thrombin uptake. Both assays were used to estimate the amount of heparin activity on a series of catheters coated with no heparin, covalently bound heparin, and ionically bound heparin. The kinetic assay produced estimates of surface-bound heparin activity and showed that some binding methods resulted in destruction of most of the heparin's biologic activity. In contrast, the thrombin uptake assay did not correlate with the amount of heparin activity on the catheter surface. Substantial thrombin uptake was found on surfaces coated with no heparin or inactive heparin, while low thrombin uptake was found on surfaces with high levels of heparin activity in the kinetic assay. We conclude that: 1) a kinetic assay based on the heparin accelerated inactivation of thrombin by antithrombin III can be used to estimate the amount of active heparin bound to a catheter surface, and 2) thrombin uptake studies do not correlate with heparin activity and do not predict which heparin binding method will result in the highest concentration of active heparin on the catheter surface.     

The interactions between antithrombin III,thrombin and surface immobilized heparin

The interactions between antithrombin III (ATIII), thrombin, and surface immobilized heparin were investigated. Carboxylated polystyrene modified with covalently immobilized albumin-heparin conjugate contain sites which can bind ATIII from buffer and plasma solutions. Approximately 65% of the ATIII molecules present at the heparinized surface either adsorbed from a buffer or plasma solution, exchanged with ATIII in buffer solution. The exchange between surface bound ATIII and ATIII in solution was repeated several times on the same heparinized surface. The number of binding sites that could bind and release ATIII was much higher when the heparinized surface was exposed to an ATIII containing buffer solution than to a plasma solution. The reduction in binding sites available for ATIII using plasma solutions as compared to buffer solutions could be explained by the competition of other plasma proteins with ATIII for the heparinized surface. It was observed that heparin binding proteins were able to compete with ATIII for binding to the immobilized heparin. Furthermore adsorption of proteins on the heparinized surface significantly reduced the availability of binding sites for ATIII. Exposure of thrombin to the heparinized surface resulted in thrombin activity at the surface. The thrombin activity on the heparinized surfaces was lower on surfaces with a higher ATIII concentration. The activity of surface bound thrombin was not affected by the presence of other plasma proteins. Enzymatically active thrombin molecules present at the heparinized surface were completely inactivated when the surface was exposed to a solution containing ATIII. The inactivation rate of surface bound thrombin by ATIII was higher than the rate of the uncatalyzed inactivation of thrombin in solution. Part of the Thrombin-Antithrombin III (TAT) complexes (10-20%) that were formed upon inactivation of thrombin remained bound to the heparinized surface. In general it was concluded that only the surface immobilized heparin molecules that can bind ATIII in a reversible way determine the anticoagulant properties of the surface. The mechanism of inactivation of a protease on a heparinized surface depends either on the catalytic effect of heparin on the inactivation rate of proteases by ATIII or on an increased uncatalytic inactivation due to increased concentrations of ATIII near the surface as compared to the concentration of ATIII in the bulk phase.     

Dermatan sulphate as an antithrombotic drug

Dermatan sulphate (DS) is a glycosaminoglycan which selectively catalyzes the inactivation of thrombin by Heparin Cofactor II without interacting with Antithrombin III. DS does not interact with other coagulation factors and, unlike heparin, is able to inactivate thrombin bound to fibrin or to the surface of an injured vessel. Efficacy and safety of DS have been validated in studies on thromboprophylaxis and on the anticoagulation for hemodialysis. Studies on thromboprophylaxis have been performed in "medical" patients as well as in general, orthopedic and oncological surgery. In this last setting, DS proved to be more efficacious than heparin, in the absence of excess bleeding. No statistically significant differences were observed between DS and heparin in hemodialysis. A low-molecular-weight DS,which shows a higher bioavailability after s.c. administration, has been tested in pilot studies on the treatment of venous thromboembolism with encouraging results. Two DS-containing compounds, sulodexide and, particularly, mesoglycan, have been clinically studied in a number of trials and found to be effective in the treatment of venous and arterial leg diseases.     

Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer

A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 microg/cm2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.     

A model of thrombin inactivation in heparinized and nonheparinized tubes with consequences for thrombus formation

The role of flow and mass transport in determining procoagulant concentration at the wall of synthetic and natural cylindrical blood vessels is analyzed theoretically. The model assumes steady laminar flow and considers, in addition to the fluid dynamic parameters, three rate-determining steps: production of procoagulant (thrombin) and its inactivation at the wall, as well as inactivation in the fluid bulk. The ratio of thrombin wall concentration to production rate Cw/N emerges as a critical parameter in characterizing the behavior of the tube wall. With a wall-inactivation rate typical of heparinized materials, Cw/N = 11.1 s/cm, independent of flow (shear rate) and axial position. This is significantly less than the range of Cw/N (50-500 s/cm) for which the thrombin concentration is high enough to result in significant fibrin formation and thrombosis. Hence little fibrin formation and a high degree of thromboresistance is expected for heparinized materials. Nonheparinized materials have Cw/N values above this range, which are only weakly dependent on shear rate and diameter, suggesting that flow-induced dispersion of thrombin (or other procoagulants) has limited impact on the thrombin wall concentration. These latter results appear to refute the conventional wisdom that attributes the relative patency of large-diameter vessels and differences between venous and arterial thrombi to such flow effects. It is likely that additional factors such as flow pulsatility and wall geometry must be considered to account for these observations.     

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.     

Chronic performance of polyurethane catheters covalently coated with ATH complex: a rabbit jugular vein model

Covalent complexes of antithrombin (AT) and heparin (ATH) have superb anticoagulant activity towards thrombin and factor Xa. Stability of polyurethane central venous catheters covalently modified with radiolabeled ATH was studied using a roller pump with saline or protease P-5147. Saline wash removed loosely bound ATH molecules to decrease graft density from 26 to 12 pmol/cm2. However, only slightly more ATH was removed by strong protease (from 12 to 7 pmol/cm2). To evaluate ATH-coated, heparin-coated, and uncoated catheters, a chronic rabbit jugular vein model was developed with catheters maintained for up to 30-106 days. Lumen occlusion was tested by drawing blood twice daily. Although unmodified or heparin-coated catheters occluded within 5-7 days after insertion, all ATH catheters remained patent throughout the experiment. Scanning electron microscopy (SEM) analysis of heparin and uncoated catheters revealed extensive thrombosis (lumen+mural) while ATH catheters were unaffected. Visual observation showed significant deposition of protein and cells on control and heparin-modified catheters and, to a lesser degree, on ATH-coated surfaces. SEM showed no fibrin inside or outside of ATH catheters, which remained patent in extended studies out to 106 days. Although atomic force microscopy showed ATH coatings to be rough, 6-fold higher anti-factor Xa activity likely contributed to increased patency. Our data confirm that ATH-modified catheters are stable and have superior potency compared to heparin or control catheters.     

Heparin cofactor II assay. Elimination of heparin and antithrombin-III effects

Functional assays for heparin cofactor II (HC-II) are based on the inactivation of thrombin by HC-II in the presence of dermatan sulfate (DS). Residual thrombin is measured in a chromogenic assay. Interference by the antithrombin-III (AT-III)/heparin complex, which also rapidly inactivates thrombin, must be eliminated from the HC-II test system. Commercial DS is contaminated with heparin, while plasma specimens to be tested contain AT-III. After NaNO2/acetic acid treatment of DS (to inactivate heparin), there was enough residual heparin to cause AT-III interference. Treatment of plasma with commercially available anti-AT-III antiserum largely, but not completely, removed AT-III interference from the HC-II assay. With commercially available reagents, both NaNO2/acetic acid treatment of DS and anti-AT-III treatment of plasma were needed to eliminate heparin/AT-III interference. Protamine sulfate inactivated DS as well as heparin and could not be used to reduce AT-III/heparin interference with the HC-II assay.     

Thrombin uptake and inhibition on endothelium and surfaces with a stable heparin coating: a comparative in vitro study

The endothelial lining and two differently heparin-coated surfaces were compared in vitro regarding thrombin uptake and inhibition. One heparin surface was based on stabilized ionic binding of heparin, the other on covalent binding of partially degraded heparin. Both heparin surfaces have previously been shown to have pronounced thromboresistant properties. The two heparinized polyethylene surfaces and the endothelial surface of segments of the porcine aorta were studied. After exposure to the surfaces, thrombin disappeared from the solution and appeared bound to the surfaces. The disappearance rate of thrombin from the solution was the same on exposure to the endothelium and the covalently bonded heparin surface, but less following exposure to the ionically bonded heparin surface. The thrombin activity appearing on the endothelium was lower than on the heparin surfaces, indicating that the endothelium exerted a slow thrombin inhibiting capacity. On exposure of the thrombin-loaded endothelium to plasma, thrombin was rapidly inhibited. Thrombin bound to the covalently bonded heparin surface was inhibited at a slower rate than on the ionically bonded surface, but still faster than the rate at which free thrombin was inhibited in nonheparinized plasma. It is concluded that the endothelium and stabilized heparin coatings bind thrombin and accelerate its inhibition by plasma.     

In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.     

Development of a temperature sensitive drug release system for polymeric implant devices

A low density polyethylene (LDPE) model surface was coated with poly(amino-p-xylylene) (amino-ppx) via chemical vapour deposition (CVD) polymerization. The functional surface was used to immobilize a polymeric drug release system consisting of poly(N-isopropylacrylamide) (NIPAAm)-co-poly(acrylic acid) (AAc). The coupled drug release system was used to incorporate the thrombin inhibitor r-hirudin. For the investigation of the concentration of incorporated r-hirudin and the release profile over a given period of time r-hirudin was labelled with 123I as well as fluorescein isothiocyanate (FITC). Using an incubation solution concentration of 0.2% at pH 5 and an ionic strength of 0.7 M a maximum concentration of 2.21 (+/- 0.11) nmol/cm2 of r-hirudin was detected. FITC-r-hirudin was almost quantitatively (2.08 +/- 10 nmol/cm2; 94%) released from the surface coating within a period of 14 days.     

In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.     

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.     

Activity toward thrombin-antithrombin of heparin immobilized on two hydrogels

Commercially obtained (Diosynth) heparin was covalently bonded to poly (vinyl alcohol) (PVA) hydrogels and to polyethylene oxide (PEO) hydrogels activated by tresyl chloride. We found that as tresyl chloride activation of PVA increased, the specific activity of the bound heparin toward thrombin and antithrombin decreased by nearly a factor of 10 and that commercial heparin bound to PEO had nearly ten-fold greater activity than when bound to PVA at comparable concentrations. These findings suggest that the long 'leash' provided by PEO hydrogels may give the heparin more access to the thrombin-antithrombin pair than the tight bond to PVA, and that crowding of heparin units on a surface limits access of the thrombin-antithrombin pair.     

Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allylamine coating for improved biocompatibility of vascular devices

The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565?±?20?ng/cm(2) of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I(2) (PGI(2)). In?vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents.     

Measurement of the rate of thrombin production in human plasma in contact with different materials.

Thrombin production in plasma in contact with various materials was consistent with a first-order autocatalytic model (d[T]/dt = kp[T]; [T] = thrombin concentration, t = time, kp = thrombin production rate constant) since the initial portion of a semilogarithmic plot of thrombin concentration against time was linear. Thrombin concentration was measured in clotting plasma (phospholipid enhanced or platelet-rich plasma) using a fluorogenic substrate (BMCA) by aliquot sampling at various intervals or more conveniently by monitoring cumulative fluorescence. The latter was generated by the action, on BMCA incubated in the clotting plasma, of the thrombin as it was generated. The thrombin concentration was determined from the first derivative of the S-shaped cumulative fluorescence curve. kp was greater for glass (7.92 x 10(-3) cm/s) than for the other materials (polypropylene, polystyrene, polyethylene and PVA; kp approximately 3.1 x 10(-3) cm/s) in plasma with cephalin without flow. A kp for heparin-PVA could not be determined since the thrombin concentration was too low to be quantified. A larger difference between polyethylene and PVA was noted with platelet-rich plasma without flow while lower values (1.0 x 10(-3) cm/s) were noted in a flow system but at a higher surface to volume ratio. The first-order rate constant can be used in simple models relating production of thrombin at a wall of a tube to its mass transfer away from the wall in flowing blood. One such model predicts that the concentration of thrombin at the wall should become infinite at the point in the tube when the mass transfer coefficient equals kp. According to this model, kp on the order of 10(-4) cm/s would be a useful target for a nonthrombogenic material.     

Involvement of thrombin in activation of heparin secretion by mast cells in immobilization stress in rats

Activation of heparin secretion by connective tissue mast cells under conditions of immobilization stress is determined by activation of the sympathoadrenal system, secretion of adrenocorticotropic hormone, and possible generation of thrombin. Generation of thrombin in the blood under these conditions is confirmed by a significant drop in the proenzyme protein C concentration by 23%, a decrease in the activity of factor V (substrate of protein C) by 36%, and prolongation of activated partial thromboplastin time by 40%. It is shown that 30-min immobilization leads to a 3-fold depletion of the heparin pool in mast cells. Intravenous injection of hirudin, a specific thrombin inhibitor, before immobilization slightly diminishes the stimulating effect of stress on heparin secretion. These data suggest that apart from catecholamines and adrenocorticotropic hormone, thrombin generated in the bloodstream during stress also markedly contributes to activation of heparin secretion by mast cells. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 2, pp. 143–145, February, 1997     

Heparinized styrene-butadiene-styrene elastomers.

A heparinized high-strength elastomer has been developed which is potentially useful as a nonthrombogenic vascular prosthesis. A surface hydroxylated styrene-butadiene-styrene (SBS) block copolymer with at least 40% extent of reaction after glow-discharge cleaning was coated with a 20% acetylated polyvinyl alcohol/heparin mixture containing glutaraldehyde and magnesium chloride. After curing at 80 degrees C for 100 min, the polyvinyl alcohol, heparin, and hydroxylated SBS were covalently bound to each other by acetal bridges. The effects of the various substrate and coating parameters were optimized to achieve very strong adhesion between the coating layer and the surface hydroxylated SBS. Heparin was not leached from the surface of the new material using 3M saline at pH 7.4 despite a detection limit of 10(-5) micrograms heparin/cm2 min. Prolonged partial thromboplastin times of greater than 1200 sec were observed (control: PTT = 120 sec). Preliminary ex vivo testing using a simple arteriovenous shunt in the leg of a rabbit showed good thromboresistance. The heparinized SBS shunt chamber remained patent for more than two hours without desorption of heparin. It was concluded that surface hydroxylated SBS heparinized by acetal coupling owed its thromboresistance to the heparin covalently bound to the surface and not to a microenvironment of heparin in solution at the blood/material interface.