Cellulophaga algicola alginate lyase inhibits biofilm formation of a clinical Pseudomonas aeruginosa strain MCC 2081.

Alginate lyases are potential agents for disrupting alginate-rich Pseudomonas biofilms in the infected lungs of cystic fibrosis patients but there is as yet no clinically approved alginate lyase that can be used as a therapeutic. We report here the endolytic alginate lyase activity of a recombinant Cellulophaga algicola alginate lyase domain (CaAly) encoded by a gene that also codes for an N-terminal carbohydrate-binding module, CBM6, and a central F-type lectin domain (CaFLD). CaAly degraded both polyM and polyG alginates with optimal temperature and pH of 37°C and pH 7, respectively, with greater preference for polyG. Recombinant CaFLD bound to fucosylated glycans with a preference for H-type 2 glycan motif, and did not have any apparent effect on the enzyme activity of the co-associated alginate lyase domain in the recombinant protein construct, CaFLD_Aly. We assessed the potential of CaAly and other alginate lyases previously reported in published literature to inhibit biofilm formation by a clinical strain, Pseudomonas aeruginosa MCC 2081. Of all the alginate lyases tested, CaAly displayed most inhibition of in vitro biofilm formation on plastic surfaces. We also assessed its inhibitory ability against P. aeruginosa 2081 biofilms formed over a monolayer of A549 lung epithelial cells. Our study indicated that CaAly is efficacious in inhibition of biofilm formation even on A549 lung epithelial cell line monolayers.

Essential role and therapeutic targeting of the glomerular endothelial glycocalyx in lupus nephritis.

Lupus nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx played central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted homing. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs and may be therapeutically targeted for SLE complications, including LN.

Topical application of Heparanase-1 facilitates bone remodeling during the healing of bone defects in a mouse model.

BACKGROUND: Although previous studies have suggested a stimulatory role of heparanase in physiological bone turnover, the potential therapeutic role of heparanase in bone healing has not been elucidated. The purpose of this study was to assess the effect of topical application of heparanase-1 on bone healing. METHODS: Two different dosages of recombinant mouse heparanase-1 and vehicle control were prepared and delivered via an osmotic pump to provide continuous topical infusion of the therapeutic reagent in a mouse bone defect model at the distal femoral metaphysis. The bone healing progress was evaluated by micro-computed tomography and histological examination at 7, 14, and 21 days after the bone defect was created. RESULTS: The peak of trabecular bone generation was achieved earlier than anticipated with the use of heparanase as measured by medullary bone volume fraction and trabecular number observed in micro-computed tomography, while the remodeling of trabecular bone to cortical bone was also achieved earlier than anticipated with the use of heparanase as measured by connectivity density. Histopathological observation revealed a higher frequency of the presence of cartilaginous tissue in the heparanase-treated groups. Both bone mineral density and cortical bone volume fraction showed the best healing outcome with low-dose heparanase, implying a biphasic effect of its mode of action. CONCLUSION: These results indicated that with the appropriate dose of topical heparanase-1, the progress of bone healing could be accelerated in vivo.

Green Chemistry Extractions of Carotenoids from Daucus carota L.-Supercritical Carbon Dioxide and Enzyme-Assisted Methods.

Multiple reviews have been published on various aspects of carotenoid extraction. Nevertheless, none of them focused on the discussion of recent green chemistry extraction protocols, especially for the carotenoids extraction from Daucus carota L. This group of bioactive compounds has been chosen for this review since most of the scientific papers proved their antioxidant properties relevant for inflammation, stress-related disorders, cancer, or neurological and neurodegenerative diseases, such as stroke and Alzheimer's Disease. Besides, carrots constitute one of the most popular sources of carotenoids. In the presented review emphasis has been placed on the supercritical carbon dioxide and enzyme-assisted extraction techniques for the relevant tetraterpenoids. The detailed descriptions of these methods, as well as practical examples, are provided. In addition, the pros and cons of each method and comparison with the standard solvent extraction have been discussed.

Alginate lyase immobilized chitosan nanoparticles of ciprofloxacin for the improved antimicrobial activity against the biofilm associated mucoid P. aeruginosa infection in cystic fibrosis.

Dense colonization of mucoid Pseudomonas aeruginosa within the self-secreted extracellular matrix (mainly alginate), called biofilm, is a principal reason for the failure of antimicrobial therapy in cystic fibrotic patients. Alginate is a key component in the biofilm of mucoid P. aeruginosa and responsible for surface adhesion and stabilization of biofilm. To overcome this problem, alginate lyase functionalized chitosan nanoparticles of ciprofloxacin were developed for the effective treatment of P. aeruginosa infection in cystic fibrosis patients. The developed nanoparticles were found to have desired quality attributes and demonstrated sustained release following the Higuchi release kinetics. Drug compatibility with the chitosan was confirmed by FTIR while powder X-ray diffraction analysis confirmed the entrapment of drug within the nanoparticle matrix. Lactose adsorbed NPs showed promising aerodynamic property. Nanoparticles showed prolonged MIC and significant reduction in biofilm aggregation and formation in planktonic bacterial suspension. Nanoparticles exhibited significantly higher inhibitory effect against biofilm of P. aeruginosa and reduced the biomass, thickness and density confirmed by confocal microscopy. Furthermore, developed nanoparticles were haemocompatible and did not exhibit any toxicity in vitro MTT assay and in vivo on lungs male Wistar rats. The data in hand collectively suggest the proposed strategy a better alternative for the effective treatment of cystic fibrosis infections.

Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase.

Although in recent years several methods have been studied and developed to obtain different types of nanosized drug delivery systems, the set up of suitable procedures and materials remains highly expensive, their preparation is time consuming and often not feasible for a scale-up process. Furthermore, the sterilisation and storage of nanocarrier formulations represents a complicated but mandatory step for their effective use. In our previous work we assessed the use of an autoclaving process to achieve, in one simple step, sterile self-assembled hyaluronan-cholesterol (HA-CH) and hyaluronan-riboflavin (HA-Rfv) nanohydrogels (NHs). In the present work, the effect of the high temperature on HA-CH has been studied in detail. HA-CH suspensions were characterised in terms of size and polydispersity by Dynamic Light Scattering at different temperatures and conditions; the HA-CH chemical structure and its molecular weight were assessed via FT-IR and GPC analysis after the sterilising cycle in an autoclave at 121°C for 20min. The obtained NHs were then observed with TEM and AFM microscopy, in both dry and liquid conditions. The Young's modulus of the NHs was determined, evidencing the soft nature of these nanosystems; the critical aggregation concentration (c.a.c) of the nanosuspension was also assessed. Thereafter, alginate lyase (AL) was conjugated to NHs, with the aim of developing a useful system for therapies against bacterial infections producing alginate biofilms. The conjugation efficiency and the enzymatic activity of AL were determined after immobilisation. The AL-NHs system showed the ability to depolymerise alginate, offering an opportunity to be a useful nanosystem for the treatment of biofilm-associated infections.

Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common joint disease that causes disabilities in elderly. However, few agents with high efficacy and low side effects have been developed to treat OA. In this study, we evaluated the effects of the alginate extract named CTX in OA cell and rabbit models. RESULTS: CTX was formulated by hydrolyzing sodium alginate polymers with alginate lyase and then mixing with pectin. HPLC was used to analyze the CTX content. Human chondrosarcoma SW1353 cells treated with interleukin-1ß were used as OA model cells to investigate the effects of CTX on chondrocyte inflammation and anabolism. CTX at concentrations up to 1000 µg/ml exerted low cytotoxicity. It inhibited the gene expression of proinflammatory matrix metalloproteinases (MMPs) including MMP1, MMP3 and MMP13 in a dose-dependent manner and increased the mRNA level of aggrecan, the major proteoglycan in articular cartilage, at 1000 µg/ml. Thirteen-week-old New Zealand White rabbits underwent a surgical anterior cruciate ligament transection and were orally treated with normal saline, glucosamine or CTX for up to 7 weeks. Examinations of the rabbit femur and tibia samples demonstrated that the rabbits taking oral CTX at a dosage of 30 mg/kg/day suffered lesser degrees of articular stiffness and histological cartilage damage than the control rabbits. CONCLUSIONS: The gene expression profiles in the cell and the examinations done on the rabbit cartilage suggest that the alginate extract CTX is a pharmaco-therapeutic agent applicable for OA therapy.

Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection.

BACKGROUND: Presence of capsule enhances the virulence of bacteria that cause pneumonia, meningitis, cystic fibrosis, dental caries, periodontitis. Capsule is an important virulence factor for Klebsiella pneumoniae and infections due to this pathogen have been associated with high mortality rates. In the present study, use of an Aeromonas punctata derived capsule depolymerase against K. pneumoniae, to reinstate the efficacy of gentamicin during pneumonia and septicemia was investigated. METHODS: Depolymerase was administered in mice intraperitoneally (50 µg) alone as well in combination with gentamicin (1.5 mg/kg), 24 h post infection during acute lung infection and 6 h later during septicemia. Bacterial load, neutrophil infiltration and cytokine levels were estimated. The immunogenicity of protein was also studied. RESULTS: In comparison to groups treated with gentamicin alone, combination treatment with depolymerase and gentamicin significantly reduced (P < 0.01) bacterial titer in the lungs, liver, kidney, spleen and blood of experimental animals. Highly significant reduction in neutrophil infiltration and levels of pro-inflammatory and anti-inflammatory cytokines was also observed. This indicated an efficient capsule removal by the enzyme, that improved gentamicin efficacy in vivo. Although the enzyme was found to be immunogenic, but no significant reduction in treatment efficacy was observed in the preimmunized as well as naïve mice. In addition, as confirmed through flow cytometry, the hyperimmune sera raised against the enzyme did not neutralize its activity. CONCLUSION: The results confirm that administration of enzyme 'depolymerase' along with gentamicin not only checked the virulence of K. pneumoniae in vivo but it also increased its susceptibility to gentamicin at a lower concentration. Such a strategy would help to avoid exposure to higher concentration of gentamicin. Moreover, since this decapsulating protein does not possess a lytic activity therefore there would be no chances of development of bacterial resistance against it. Therefore, it should be studied further for its successful inclusion in our prophylactic/therapeutic regimes.

Effect of intradermal injection of heparinase III on skin wound healing in diabetic rats.

AIM: neuropathy and vascular damage in this disease. Heparanase is an endoglycosidase that degrades heparan sulfate in the extracellular matrix and is believed to promote angiogenesis. The present study has been performed to investigate the effect of heparinase III (an enzyme which exclusively cleaves heparan sulfate) on wound healing in diabetic rats. METHODS: The rats became diabetic by a single streptozotocin injection. Two weeks later, a wound was created by excision of the skin in the left paravertebral area. Heparinase III (0.2 unit) was injected intradermally around the wound every 5 days, starting on day one, for a total of three doses. The wound area was measured every 3 days. After completion of wound healing, full thickness skin samples were taken from the wound sites and evaluated for volume density of the collagen bundles, numerical density of the fibroblasts, and length density of the vessels. RESULTS: Heparinase III accelerated wound closure compared to control diabetic animals. Microscopical examination revealed that it increased angiogenesis with no significant effect on collagen density and the number of fibroblasts. CONCLUSION: Heparinase III induces angiogenesis and improves wound healing in diabetic animal model.

Fractone-heparan sulphates mediate FGF-2 stimulation of cell proliferation in the adult subventricular zone.

OBJECTIVES: Fractones are extracellular matrix structures that form a niche for neural stem cells and their immediate progeny in the subventricular zone of the lateral ventricle (SVZa), the primary neurogenic zone in the adult brain. We have previously shown that heparan sulphates (HS) associated with fractones bind fibroblast growth factor-2 (FGF-2), a powerful mitotic growth factor in the SVZa. Here, our objective was to determine whether the binding of FGF-2 to fractone-HS is implicated in the mechanism leading to cell proliferation in the SVZa. MATERIALS AND METHODS: Heparitinase-1 was intracerebroventricularly injected with FGF-2 to N-desulfate HS proteoglycans and determine whether the loss of HS and of FGF-2 binding to fractones modifies FGF-2 effect on cell proliferation. We also examined in vivo the binding of Alexa-Fluor-FGF-2 in relationship with the location of HS immunoreactivity in the SVZa. RESULTS: Heparatinase-1 drastically reduced the stimulatory effect of FGF-2 on cell proliferation in the SVZa. Alexa-Fluor-FGF-2 binding was strictly co-localized with HS immunoreactivity in fractones and adjacent vascular basement membranes in the SVZa. CONCLUSIONS: Our results demonstrate that FGF-2 requires HS to stimulate cell proliferation in the SVZa and suggest that HS associated with fractones and vascular basement membranes are responsible for activating FGF-2. Therefore, fractones and vascular basement membranes may function as a HS niche to drive cell proliferation in the adult neurogenic zone.

Impact of the composition of alginate and gelatin derivatives in bioconjugated hydrogels on the fabrication of cell sheets and spherical tissues with living cell sheaths.

Gelatin and alginate derivatives possessing phenolic hydroxyl moieties (gelatin-Ph and Alg-Ph) were dissolved in aqueous solution and conjugated via horseradish peroxidase-catalyzed crosslinking, resulting in hydrogelation. The objective of creating the hydrogels was to prepare cell sheets and spherical tissues wrapped in living cell sheaths. An increase in the gelatin-Ph content in the hydrogel improved cellular adhesion on the hydrogel surface but hindered degradability by alginate lyase. A hydrogel with the desired characteristics was obtained from a solution containing 0.5% (w/v) gelatin-Ph and 1.5% (w/v) Alg-Ph. Human aortic endothelial (HAE) cells and mouse embryo fibroblast 10T1/2 cells grew on the hydrogels and could be harvested as cell sheets by treatment with alginate lyase. 10T1/2 cells enclosed in Alg-Ph/gelatin-Ph microcapsules composed of the conjugate hydrogel elongated on the inner surface of the microcapsules and grew three times faster than those enclosed in Alg-Ph microcapsules. Alg-Ph/gelatin-Ph microcapsules not only supported growth of the enclosed cells into spherical tissues, but also provided a cell adhesive outer surface for the fabrication of an HAE cell layer. Finally, spherical tissues of 10T1/2 cells wrapped in living HAE cell sheaths were obtained by treatment with alginate lyase.

Glycosidic enzymes enhance retinal transduction following intravitreal delivery of AAV2.

PURPOSE: To determine whether the co-injection of extracellular matrix degrading enzymes improves retinal transduction following intravitreal delivery of adeno-associated virus-2 (AAV2). METHODS: AAV2 containing cDNA encoding enhanced green fluorescent protein (GFP), under the control of a chicken ß-actin promoter, was delivered by intravitreal injection to adult mice in conjunction with enzymes including collagenase, hyaluronan lyase, heparinase III, or chondroitin ABC lyase. Two weeks later, retinal flatmounts were examined for GFP expression using confocal microscopy. RESULTS: Without the addition of enzymes, transduction was limited to occasional cells in the retinal ganglion cell layer. The addition of heparinase III or chondroitin ABC lyase greatly enhanced transduction of the retinal ganglion cell layer and increased the depth of transduction into the outer retina. Hyaluronan lyase had a limited effect and collagenase was ineffective. Electroretinograms survived with higher concentrations of heparinase III and chondroitin ABC lyase than were required for optimal retinal transduction. CONCLUSIONS: AAV2-mediated retinal transduction is improved by co-injection of heparinase III or chondroitin ABC lyase. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to both the inner and outer retina.

Statistical optimization of single-cell production from Taxus cuspidata plant cell aggregates.

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200 × g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.

Degradation of the endothelial glycocalyx is associated with chylomicron leakage in mouse cremaster muscle microcirculation.

A thick endothelial glycocalyx contributes to the barrier function of vascular endothelium in macro- and microcirculation. We hypothesised in the current study that diet-induced hyperlipidaemia perturbs the glycocalyx, resulting in decreased dimensions of this layer and increased transendothelial lipoprotein leakage in capillaries. Glycocalyx thickness was measured in mouse cremaster muscle capillaries by intravital microscopy from the distance between flowing red blood cells and the endothelial surface. In control C57BL/6 mice on standard chow, glycocalyx thickness measured 0.58 ± 0.01 (mean ± SEM) µm, and no lipoproteins were observed in the tissue. After three months administration of an either mild or severe high-fat / high-cholesterol diet (HFC) to C57BL/6 and ApoE3-Leiden mice, circulating large lipoproteins appeared into the subendothelial space in an increasing proportion of cremaster capillaries, and these capillaries displayed reduced glycocalyx dimensions of 0.40 ± 0.02 and 0.30 ± 0.01 µm (C57BL/6 mice), and 0.37 ± 0.01 and 0.28 ± 0.01 µm (ApoE3-Leiden mice), after the mild and severe HFC diet, respectively. The chylomicron nature of the accumulated lipoproteins was confirmed by observations of subendothelial deposition of DiI-labeled chylomicrons in capillaries after inducing acute glycocalyx degradation by heparitinase in normolipidaemic C57BL/6 mice. It is concluded that while under control conditions the endothelial glycocalyx contributes to the vascular barrier against transvascular lipoprotein leakage in the microcirculation, diet-induced hyperlipidaemia reduces the thickness of the glycocalyx, thereby facilitating leakage of chylomicrons across the capillary wall.

[Use of hyaluronic acid cleaving enzymes for absorption acceleration. Results of an in vitro study with xylazine and ketamine]. / Einsatz von Hyaluronsäure spaltenden Enzymen zur Resorptionsbeschleunigung. Ergebnisse einer In-vitro-Studie mit Xylazin und Ketamin.

The so-called "Hellabrunner Mischung", (combination of xylazine and ketamine with hyaluronidase) is frequently used for the immobilisation of wildlife animals. The enzyme hyaluronidase shall improve the distribution of the intramuscularly or subcutaneously administered compounds in the tissue and enhance their absorption. These enhancing effects of two hyaluronate lyases of bacterial origin (Streptococcus agalactiae and Streptococcus equisimilis) and a testicular hyaluronidase were compared in an in vitro test. Using the isolated perfused bovine udder, 2 ml of a solution were administered subcutaneously containing 125 mg/ml xylazine and 100 mg/ml ketamine and one of the above mentioned enzymes (150 I.U.). All three enzymes enhanced the absorption rate of xylazine and ketamine determined by measurement of the concentration in the perfusate. The bacterial hyaluronate lyases were significantly more efficient, especially during the clinically important first minutes after administration.

Endothelial cell glycocalyx modulates immobilization of leukocytes at the endothelial surface.

OBJECTIVE: A thick endothelial glycocalyx provides the endothelial surface with a nonadherent shield. Oxidized LDL (Ox-LDL) degrades the endothelial glycocalyx. We hypothesized that glycocalyx degradation stimulates leukocyte-endothelial cell adhesion, whereas intravascular supplementation with sulfated polysaccharides reconstitutes the endothelial glycocalyx and attenuates Ox-LDL-induced leukocyte-endothelial cell adhesion. METHODS AND RESULTS: Degradation of the endothelial glycocalyx by local microinjection of heparitinase (10 to 50 U/mL) into mouse cremaster venules dose-dependently increased the number of adherent leukocytes. Systemic administration of Ox-LDL (0.4 mg/100 g body weight) induced 10.1+/-0.9 adherent leukocytes/100 microm at 60 minutes. In the venules perfused with 500-kDa dextran sulfate (1 mg/mL), the number of adherent leukocytes at 60 minutes after Ox-LDL bolus application was not influenced (9.2+/-1.0 leukocytes/100 microm). However, the venules locally perfused with heparan sulfate (10 mg/mL) or heparin (1 mg/mL) displayed a significantly lower number of adherent leukocytes induced by Ox-LDL: 5.1+/-0.7 and 5.4+/-0.9 leukocytes/100 microm, respectively (P<0.05). Fluorescently labeled heparan sulfate and heparin, but not dextran sulfate, attached to the venule luminal surface after Ox-LDL administration. CONCLUSIONS: Endothelial glycocalyx degradation stimulates leukocyte immobilization at the endothelial surface. Circulating heparan sulfate and heparin attach to the venule wall and attenuate Ox-LDL-induced leukocyte immobilization.

Cellular mechanisms of heparinase III protection in rat traumatic shock.

Pentobarbital-anesthetized rats subjected to traumatic shock developed a shock state characterized by marked hypotension to 65-70 mmHg, a survival time of 88 +/- 13 min, significant increases in ileal myeloperoxidase activity (P < 0.01), and severe endothelial dysfunction as evidenced by a significant (P < 0.01) decrease in vasorelaxation to endothelium-dependent dilators. Treatment with heparinase III (45 microg . kg-1 . min-1) 10 min posttrauma prolonged survival time to 223 +/- 19 min (P < 0.001), significantly attenuated ileal myeloperoxidase activity (P < 0.01), and significantly preserved endothelial function (P < 0.05). Intravital microscopy of the rat mesentery showed that infusion of heparinase III (45-67 microg . kg-1 . min-1) significantly (P < 0.01) attenuated both leukocyte rolling and adherence in the rat mesenteric microvasculature in response to NG-nitro-L-arginine methyl ester stimulation. Immunohistochemical localization of surface-expressed P-selectin on mesenteric venules showed that heparinase III infusion at 45-67 microg . kg-1 . min-1 significantly (P < 0.05) attenuated the increase in surface P-selectin expression. The beneficial effects of heparinase III are mediated at least in part by attenuating leukocyte-endothelial cell interactions via a P-selectin-dependent mechanism.

A comparison of thromboelastography with heparinase or protamine sulfate added in vitro during heparinized cardiopulmonary bypass.

Thromboelastography (TEG) has been used after cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage. Conventional TEG during CPB is not possible due to the sensitivity of the TEG to even small amounts of heparin, which produces a nondiagnostic tracing. The purpose of this study was to compare heparin neutralization using heparinase or protamine in TEG blood samples obtained during CPB. TEG testing was performed on 48 patients before, during and after CPB. Tissue plasminogen activator activity and antigen were measured on a subset of 32 patients. We found: 1) heparinase neutralized at least 10 IU/ml heparin while 1.6 ug/ml protamine neutralized up to 7 IU/ml heparin, 2) in samples with complete heparin neutralization by both methods, there was no significant difference in the R values, 3) while there was good correlation for other TEG parameters between heparinase and protamine treated samples, heparinase treatment produced shorter K values and higher angle, MA and A60, 4) while fibrinolysis was detected using both methods, heparinase treatment suppressed fibrinolysis in the TEG in both samples from patients and after in vitro addition of tissue plasminogen activator, 5) TEG was not a sensitive indicator of t-PA activity, detecting only 21% of samples with increased t-PA activity during bypass, and 5) heparinase was at least 100 times more expensive than protamine. We conclude that while both heparinase and protamine can be used to neutralize heparin in TEG samples obtained during CPB, protamine neutralization is more sensitive to fibrinolysis and less expensive, but the protamine dose must be carefully selected to match the heparin level used at individual institutions.

Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans.

Heparan sulfate proteoglycans (HSPG) are involved in the binding and uptake of apolipoprotein (apo) E-enriched remnant lipoproteins by cultured cells in vitro. To define the role of hepatic HSPG in remnant lipoprotein clearance in vivo, heparinase (30 units) was infused intravenously into mice to hydrolyze the liver HSPG and determine the effect of HSPG hydrolysis on remnant clearance by the liver. Liver HSPG were prelabeled by peritoneal injection of [35S]Na2SO4. Injection of heparinase decreased the amount of 35S-labeled liver HSPG by approximately 20-40% within 10-15 min. Heparinase infusion significantly inhibited the clearance of chylomicrons, chylomicron remnants, chylomicron remnants + apoE, rabbit beta-very low density lipoproteins (beta-VLDL), and beta-VLDL + apoE. Compared with saline injection in control mice, heparinase injection retarded the plasma clearance of the remnants by 1.5- to 2-fold and decreased liver uptake by 1.3- to 1.6-fold. Confocal fluorescence microscopy of thick slices of liver from mice injected with 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine-labeled beta-VLDL + apoE revealed markedly less intense fluorescence from hepatocytes in heparinase-treated animals compared with those in saline-treated control animals. Intravenous heparinase infusion did not inhibit the clearance of mouse low density lipoproteins (LDL), a ligand for the LDL receptor, and did not affect the clearance of alpha 2-macroglobulin, a ligand for the LDL receptor-related protein. The results suggest an important role of the liver HSPG in remnant clearance in vivo.

Heparinase: in vivo activity and immunogenicity in rabbits.

Anticoagulation with heparin is required during extracorporeal circulation for hemodialysis and cardiopulmonary bypass as well as during vascular surgery. Reversal of anticoagulation with protamine may be associated with hypotension and rebound anticoagulation and requires stoichiometric doses. Heparinase from Flavobacterium heparinum catalytically degrades heparin and reverses its anticoagulant effect. Heparin was administered to New Zealand White rabbits and plasma levels were assayed with the APTT anticoagulant assay and the azure A chemical assay. Heparinase actively degraded heparin both in vitro in rabbit plasma and in vivo in rabbit blood as determined by both the anticoagulant and chemical assays when compared to control heparin disappearance curves. Antibodies to heparinase were demonstrated by the ELISA technique in rabbits receiving i.v. heparinase. These antibodies, however, did not effect the activity of the enzyme in vitro or in vivo. No toxic effects of heparinase were noted in observations of the animals or in blood and histologic studies. Heparinase, either free or immobilized, may be a useful heparin-reversing agent without the drawbacks of protamine.