Antithrombin III binding to surface immobilized heparin and its relation to F Xa inhibition.

The mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depolymerized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 and 11 picomoles/cm2, respectively. Thus the estimated density of biologically active high and low affinity heparin respectively would be 40 and 90 ng/cm2. The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. It was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromised AT binding sequences.

A new model to assess staphylococcal adhesion to intraocular lenses under in vitro flow conditions.

Adhesion of staphylococcal cells to intraocular lenses coated with heparin was studied under in vitro flow conditions (280 microl min(-1)) at 37 degrees C. The intraocular lenses were incubated with human cerebrospinal fluid for 1 h or with cerebrospinal fluid including 0.50% plasma for 12 h, prior to bacterial challenge. Two strains of Staphylococcus epidermidis selected for this study, were isolated from biomaterial-associated infections. Bacterial adhesion was quantitated by bioluminescence and visualized by fluorescence microscopy of acridine orange stained bacteria. Surface coating with heparin significantly decreased bacterial adhesion of both strains after incubation with cerebrospinal fluid including 0.50% plasma for 12 h (p = 0.0209). However, no difference in bacterial adhesion was obtained between intraocular lenses with and without heparin, after incubation with cerebrospinal fluid for 1 h (p = 0.327). Microscopy showed that more bacteria were present on intraocular lenses without heparin than on intraocular lenses with heparin. The results show that preincubation with a proteinaceous fluid influences subsequent bacterial adhesion to the polymer surface. The results suggest that IOLs with heparin coating may be less prone to bacterial adhesion under perfusion conditions after surface conditioning in human CSF with 0.50% plasma and a preincubation period of 12 h. Heparin coating might be a valuable tool to decrease implant-associated bacterial endophthalmitis.

Adhesion of coagulase-negative staphylococci and adsorption of plasma proteins to heparinized polymer surfaces.

Protease treatment of cells of coagulase-negative staphylococci reduced the adhesion of bacteria to heparinized polyethylene preadsorbed with serum. Fibronectin (Fn), fibrinogen (Fg), vitronectin, complement factor C3c, plasminogen, laminin and to a low extent albumin were detected on tridodecylmethylammonium chloride (TDMAC)-heparinized polyvinyl chloride (PVC) catheters extirpated from the circulation of patients. Using a perfusion model we show that during the first hours of perfusion with human plasma, Fn and Vn dominate, whereas after 22 h of perfusion Fg is the dominant protein. Field emission scanning electron microscopy and atomic force microscopy studies on TDMAC-heparinized catheters as well as on end-point attached heparinized PVC catheters indicate that quantitatively more Fg than Fn is exposed on these surfaces after prolonged exposure (> 22 h) to human plasma.

Helicobacter pylori interacts with heparin and heparin-dependent growth factors.

The pathogenic bacterium Helicobacter pylori, which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 x 10(-12)M), and also heparan sulfate and heparin with higher affinity (Kd 9 x 10(-9)M) than FGFs bind to heparin (10(-8) - 10(-9)M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori, which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanisms. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.

Thrombin inactivation on surfaces with covalently bonded heparin.

About 8000 Daltons porcine mucosa heparin fragments were covalently bonded by end-point attachment to polyethylene. The interaction between the immobilized heparin, added thrombin, and antithrombin III [AT] was investigated. The heparin surface was adsorbed with either albumin, AT dissolved in albumin or Tyrode, or platelet free plasma. Irrespective of the pre-treatment procedure, exposure of the surface to thrombin resulted in the same substantial decrease of thrombin in solution and the same degree of surface-confined thrombin activity. It was concluded that the heparin surface has a large capacity to bind thrombin and that the thrombin inhibitory capacity of high affinity heparin fragments is limited. On exposure of the thrombin-loaded surfaces to defibrinogenated plasma or AT, the surface-confined thrombin was inhibited within 30 seconds. Successive dilutions of plasma or AT decreased the inhibition rate but not the inhibition capacity. It is concluded that inhibition of thrombin adsorbed on the heparin surface occurs as follows: Added AT adheres to high affinity heparin fragments on the surface whereupon adsorbed thrombin migrates in the hydrophilic heparin coating towards the reaction site of AT and becomes inhibited. The inactivated thrombin-AT complex leaves then the surface, thus enabling the process to be repeated.

Mechanisms of anticoagulant effects of some sulphated polysaccharides.

The anticoagulant activity of a partially reduced sulphated alginic acid, a partially reduced aminated and sulphated alginic acid and sulphated guaran have been studied. The anticoagulant activities in the APTT assay were 28, 39 and 70 IU/mg respectively. None showed any activity in anti-factor Xa assay. Studies on binding to Antithrombin III - Sepharose showed that sulphated guaran and a fraction of the aminated and sulphated alginic acid was bound, whereas no binding occurred with sulphated alginic acid. The inhibition of thrombin activity by these polysaccharides was studied in purified systems with or without added Antithrombin III, using both fibrinogen clotting and chromogenic peptide substrate assays. The two alginic acid preparations showed Antithrombin III-dependent inhibition of thrombin, whereas the sulphated guaran inhibits both by Antithrombin III-dependent and independent mechanisms.

Bromine oxidation of methyl alpha- and beta-pyranosides of D-galactose, D-glucose, and D-mannose.

Methyl alpha- and beta-pyranosides of D-galactose, D-glucose, and D-mannose have been oxidized with bromine in aqueous solution at various pH values. The resulting keto glycosides were converted into their more-stable O-methyloxime derivatives which were characterized by spectroscopy and chromatography. Oxidation at a ring carbon atom where the hydrogen is axial is hindered by bulky substituents in syn (i.e., a 1,3) diaxial relationship. Thus, the aglycon group in the alpha anomers protects position 3, the axial HO-4 in galactopyranosides protects position 2, and the axial HO-2 in mannopyranosides protects position 4 from oxidation.

Effect in a rat model of heparinized peritoneal dialysis catheters on bacterial colonization and the healing of the exit site.

We performed a prospective, double-blind, randomized study to evaluate whether stable surface heparinization of silicone peritoneal dialysis (PD) catheters prevents bacterial colonization or biofilm formation and improves healing of the exit site. Heparinized catheters were implanted in 20 Sprague-Dawley rats (group H) and non heparinized catheters in another 20 (group C). The PD catheters, constructed of silicon tubing with two polyester cuffs, were patterned after the standard Tenckhoff catheter. A covalent multipoint method of attachment onto polymeric surfaces was used for stable, permanent chemical immobilization of heparin on the PD catheter. Dialysis exchanges (25-mL instillation volume) were performed twice daily for 4 weeks through the permanent catheter. Prophylactic antibiotics were not used. The exit sites were evaluated at 2-week intervals. The extent of biofilm coverage on the intraperitoneal portion of the catheter (obtained at the end of the experiment) was assessed, and sonicated fluid from the catheter tip was cultured for evaluating bacterial colonization of the catheter. Exit-site scores in group H were lower than in group C (p = 0.052) at the end of week 4. Bacterial colonization tended to be less common in group H [2 of 12 catheters (17%)] than in group C [8 of 15 catheters (53%); p = 0.058], but the extent of biofilm, the peritonitis rate, and the inflammation score of tissue adjacent to the cuff were not different between the groups. Those data suggest that heparinized PD catheters can be a practical approach to the prevention of bacterial colonization and can improve healing of the exit site.

Immobilised heparin accelerates the healing of human wounds in vivo.

Locally produced growth factors are of great importance in wound healing in human skin. Wound fluid from chronic wounds contains low concentrations of growth factors possibly because of rapid degradation as a result of the high concentration of proteases. Many growth factors involved in wound healing bind to heparin and are thereby stabilised and activated. We have recently shown that heparin in combination with chitosan stimulates re-epithelialisation in an in vitro model of human wound healing. In the present study we investigated the effects of a chitosan-heparin membrane on wound healing in 10 split-thickness graft donor sites in human skin. The chitosan-heparin membrane stimulated healing of the donor sites both when judged macroscopically in a blinded fashion and when biopsy specimens from the treated and untreated parts of the wound were investigated microscopically. We hypothesise that the beneficial effects of the chitosan-heparin membrane result from slow release of heparin into the wound area which protects locally produced growth factors. The result is increased stabilisation and concentration of growth factors in the wound area, which stimulate healing. We believe that these results may be important in the treatment of wounds that are reluctant to heal.

Heparin-chitosan complexes stimulate wound healing in human skin.

The effect of heparin ionically linked to chitosan on the stimulation of re-epithelialisation of full thickness wounds in human skin was investigated in an in vitro model. After seven days of incubation, heparin-chitosan gel stimulated 9/10 of the full thickness wounds to re-epithelialise compared with only 3/10 of the wounds that were covered with chitosan gel or membrane, and none of the wounds incubated without gel or membrane or with heparin solution alone. Both dermal and epidermal cells were viable after the incubation time. Furthermore, the stimulatory effect of the heparin-chitosan complexes depended on the concentration of heparin in the complex. We hypothesise that these effects are caused by stabilisation and activation of growth factors that bind to immobilised heparin.