Effect of fibronectin on the binding of antithrombin III to immobilized heparin

An objective of this research is to verify the mechanism of anticoagulant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. \ud Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. \ud The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin

Release of proteins via ion exchange from albumin-heparin microspheres

Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg lysozyme on albumin-heparin microspheres was linear until saturation was abruptly reached,\ud \ud The adsorption isotherms of human lysozyme at low and high ionic strength were typical of adsorption isotherms of proteins on ion exchange gels. The adsorption of human lysozyme on albumin-heparin and albumin microspheres fit the Freundlich equation suggesting heterogeneous binding sites. This was consistent with the proposed multivalent, electrostatic interactions between human lysozyme and negatively charged microspheres. Scatchard plots of the adsorption processes of human lysozyme on albumin-heparin and albumin microspheres suggested negative cooperativity, while positive cooperativity was observed for chicken egg lysozyme adsorption on albumin-heparin microspheres.\ud \ud Human lysozyme loading of albumin-heparin microspheres was 3 times higher than with albumin microspheres, with long term release occurring via an ion exchange mechanism. Apparent diffusion coefficients of 2.1 × 10-1 and 3.9 × 10-11cm2/sec were obtained for the release of human lysozyme from albumin-heparin and albumin microspheres, respectively. The release was found to be independent of diffusion, since the rate determining step was likely an adsorption/desorption processes. An apparent diffusion coefficient of 4.1 × 10-12 cm2/sec was determined for the release of chicken egg lysozyme from albumin-heparin microspheres.\ud \ud Low release of the lysozymes from albumin-heparin microspheres was observed in deionized water, consistent with the proposed ion exchange release mechanism. Overall, albumin-heparin microspheres demonstrated enhanced ion exchange characteristics over albumin microspheres

pH-Sensitive Chitosan–Heparin Nanoparticles for Effective Delivery of Genetic Drugs into Epithelial Cells

Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosan–heparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosan–heparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosan–heparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosan–heparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors

Heparin bonded lines offer a solution that is difficult to implement Rapid Response to Dobson,R "Half the cases of bacteraemia in hospitals in England are linked to devices)

[Full text] The BMJ news report[1] that devices, particularly central venous lines, are linked to over half the cases of hospital acquired bacteraemia has long been a source of concern to clinicians in intensive care. Unfortunately, one simple, effective and low-cost intervention is not being used because of restrictions on licensing and supply. A systematic review[2] of heparin bonding (1 trial) or heparin infusion (3 trials) versus no anti-thrombotic prophylaxis for central venous lines showed a strong trend for a reduction in bacteraemia (pooled RR 0.26; 95% CI: 007, 1.03). Our updated searches found one subsequent trial[3] (200 children), conducted at Great Ormond Street Hospital (GOSH) without commercial funding, in which heparin bonded lines reduced the risk of bacteraemia from 33% to 4% (risk difference 29%; 19%, 39%). As heparin bonded central venous lines are not licensed for use in the UK, GOSH has been able to maintain their supply only by agreeing to take full legal responsibility should problems arise. This means that lines can be used but only on a named-patient basis with their use audited. It is therefore not surprising that a recent survey of 36 consultant and specialist trainee anaesthetists from across the south east of England found that no other trust was using heparin bonded lines. Policy regarding heparin bonded lines undoubtedly requires more and better quality studies[4,5]. However, satisfactory alternatives are few. Heparin infusion requires staff time, additional equipment, and provides the opportunity for drug errors. In contrast, adverse effects of heparin bonded lines remain theoretical. Although heparin bonded lines are licensed and widely used in the USA, the UK licensing authority requires further studies which the company appears to be reluctant to pursue. Focused commercial and research efforts are required to resolve this impasse. Reference List 1. Dobson R. Half the cases of bacteraemia in hospitals in England are linked to devices. BMJ 2003;326:10. 2. Randolph AG, Cook DJ, Gonzales CA, Andrew M. Benefit of heparin in central venous and pulmonary artery catheters: a meta-analysis of randomized controlled trials. Chest 1998;113:165-71. 3. Pierce CM, Wade A, Mok Q. Heparin-bonded central venous lines reduce thrombotic and infective complications in critically ill children. Intensive Care Med. 2000;26:967-72. 4. Randolph AG, Cook DJ, Gonzales CA, Andrew M. Benefit of heparin in peripheral venous and arterial catheters: systematic review and meta- analysis of randomised controlled trials. BMJ 1998;316:969-75. 5. Shah PS, Ng E, Sinha AK. Heparin for prolonging peripheral intravenous catheter use in neonates (Cochrane Review). Cochrane Database.Syst.Rev. 2002;CD00277

The ISFET based heparin sensor with a monolayer of protamine as affinity ligand

The ion-step measuring method was used to determine absolute heparin concentrations in PBS and blood plasma with a Ta2O5 ISFET on to which a monolayer of protamine had been immobilized. Heparin is a highly negatively charged polysaccharide, which is used clinically to delay the clotting of blood. Protamine acts as an affinity ligand for heparin. The response of the ISFET system on a step-wise increase in the electrolyte concentration (a so-called ion-step) is a transient change of the output voltage, which is related to the surface charge density of the ISFET gate oxide. After 2 mins of incubation in a plasma sample containing heparin, the amplitude of the transient ISFET response to an ion-step showed a linear relation to the heparin concentration. In blood plasma, heparin concentrations between 0·3 and 2·0 Units/ml could be determined with an accuracy of ±0·08 Units/ml. Heparin concentrations in different plasma samples of heparinized patients were determined and compared with the APTT. No direct relation was found between the APTT and the heparin concentration, but this result was not surprising

Poly(dimethylsiloxane)-poly(ethyleneoxide)-heparin block copolymers. I. Synthesis and characterization

Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), and heparin (PDMS-PEO-Hep) have been prepared via a series of coupling reactions using functionalized prepolymers, diisocyanates, and derivatized heparins. All intermediate steps of the synthesis yield quantifiable products with reactive end-groups, while the final products demonstrate bioactive, covalently bound heparin moieties. Due to the solvent systems required, commercial sodium heparin was converted to its benzyltrimethyl ammonium salt to enhance its solubility. The same procedure was applied to heparin degraded by nitrous acid in order to covalently couple it in solutions with the semitelechelic copolymers. As might be expected, this derivatization reduces the apparent bioactivity of the heparin. However, preliminary findings suggest that the bioactivity can be restored by reforming the heparin sodium salt

Interaction of antithrombin III with surface-immobilized albumin-heparin conjugates

The interaction between antithrombin III (ATIII) and albumin-heparin conjugates covalently coupled onto carboxylated polystyrene beads either in buffer containing albumin or in plasma was studied using 14C-labeled ATIII. Binding isotherms of ATIII were modeled using a summation of two Langmuir equations. These equations describe the binding of ATIII to two different sets of binding sites, one with a high, the other with a low affinity for ATIII. The average binding constants for the binding of ATIII to these sites are 9 × 106 L/mol and 0.3 × 106 L/mol, respectively. The binding of ATIII to surface binding sites with a high affinity for ATIII was correlated with the presence of specific ATIII binding sites in the immobilized heparin. Binding of ATIII from albumin solutions to binding sites with a low affinity for ATIII was dominated by nonspecific binding of ATIII to the immobilized heparin. A third small fraction of the surface bound ATIII is probably adsorbed to sites on the surface not covered with heparin. In the case of the binding of ATIII to the heparinized surface from plasma solutions, a fraction of initially adsorbed ATIII was desorbed by other plasma proteins. This desorption in combination with direct competition between ATIII and other plasma proteins resulted in lower ATIII surface concentrations using plasma as compared to the ATIII surface concentrations obtained using albumin solutions. The binding of ATIII to nonspecific binding sites was almost completely inhibited in the presence of plasma proteins. The amount of ATIII bound to immobilized heparin via specific ATIII binding sites was 30% lower in plasma solutions as compared to the specific binding of ATIII using albumin solutions. It is concluded that the accessibility of immobilized heparin for ATIII in plasma decreases by binding of heparin-binding proteins onto the immobilized heparin and/or by adsorption of other plasma proteins on the heparinized surface

Heparin and air filters reduce embolic events caused by intra-arterial cerebral angiography - A prospective, randomized trial

Background-Intra-arterial cerebral angiography is associated with a low risk for neurological complications, but clinically silent ischemic events after angiography have been seen in a substantial number of patients.Methods and Results-In a prospective study, diffusion-weighted magnetic resonance imaging (DW-MRI) before and after intra-arterial cerebral angiography and transcranial Doppler sonography during angiography were used to evaluate the frequency of cerebral embolism. One hundred fifty diagnostic cerebral angiographies were randomized into 50 procedures, each using conventional angiographic technique, or systemic heparin treatment throughout the procedure, or air filters between the catheter and both the contrast medium syringe and the catheter flushing. There was no neurological complication during or after angiography. Overall, DW-MRI revealed 26 new ischemic lesions in 17 patients (11%). In the control group, 11 patients showed a total of 18 lesions. In the heparin group, 3 patients showed a total of 4 lesions. In the air filter group, 3 patients exhibited a total of 4 lesions. The reduced incidence of ischemic events in the heparin and air filter groups compared with the control group was significantly different (P=0.002). Transcranial Doppler sonography demonstrated a large number of microembolic signals that was significantly lower in the air filter group compared with the heparin and control groups (P=0.01), which did not differ from each other.Conclusions-Air filters and heparin both reduce the incidence of silent ischemic events detected by DW-MRI after intra-arterial cerebral angiography and can potentially lower clinically overt ischemic complications. This may apply to any intra-arterial angiographic procedure