Synthesis and nonthrombogenicity of polyetherurethaneurea film grafted with poly(sodium vinyl sulfonate).

Synthesis of nonthrombogenic materials without using biologically active substances was explored. Poly(sodium vinyl sulfonate) is a water-soluble synthetic polymer and activates antithrombin III to exert nonthrombogenicity that was dependent on the molecular weight. Polyetherurethaneurea film was plasma-treated and graft-polymerized with sodium vinyl sulfonate. The graft film showed excellent in vitro and ex vivo nonthrombogenicity by suppressing interactions with plasma proteins and platelets as well as by inactivating blood-clotting factors.     

Hemocompatibility of human whole blood on polymers with a phospholipid polar group and its mechanism.

The hemocompatibility of a polymer containing a phospholipid polar group, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate(BMA)), with human whole blood was evaluated. When human whole blood without an anticoagulant was contacted with polymers, the blood cell adhesion and aggregation on the polymer without the MPC moiety was extensive, and considerable fibrin deposition was observed. This phenomenon was suppressed with an increase in the polymer MPC composition. Thus, the MPC moiety in the copolymer plays an important role in the nonthrombogenic behavior of the copolymer. These results were also confirmed by the whole blood coagulation time on the polymer surface which was determined by Lee-White method. The adsorption of phospholipids and proteins from human plasma on poly(MPC-co-BMA) was investigated to clarify the mechanism of the nonthrombogenicity observed with the polymer. The amount of phospholipids was increased; whereas, adsorbed proteins were decreased with an increase in the MPC composition. From these results, we concluded that the phospholipids adsorbed on poly(MPC-co-BMA) play the most important role in the nonthrombogenicity of the MPC copolymer.     

What really is blood compatibility?

The criteria for nonthrombogenicity are classically defined as long clotting times and minimal platelet deposition. The inability to point to unequivocal progress in the development of truly nonthrombogenic materials, highlights the inadequacy if not actually invalidity of these criteria. Our approach is to define nonthrombogenicity in terms of: (1) a thrombin production rate constant, kp < 10(-4) cm s(-1); (2) low platelet consumption and low degree of platelet activation (e.g., microparticle formation); (3) perhaps some platelet spreading; and (4) low complement and leukocyte activation. Only when the target becomes clear, will it be possible to identify clear strategies for producing the materials we need.     

What really is blood compatibility?

The criteria for nonthrombogenicity are classically defined as long clotting times and minimal platelet deposition. The inability to point to unequivocal progress in the development of truly nonthrombogenic materials, highlights the inadequacy if not actually invalidity of these criteria. Our approach is to define nonthrombogenicity in terms of: (1) a thrombin production rate constant, k_p < 10^-4 cm s^-1; (2) low platelet consumption and low degree of platelet activation (e.g., microparticle formation); (3) perhaps some platelet spreading; and (4) low complement and leukocyte activation. Only when the target becomes clear, will it be possible to identify clear strategies for producing the materials we need.     

Probe of specific interaction between a simplified synthetic glycopolymer and erythrocytes as mediated by a glucose transporter (GLUT) on a cell membrane.

In order to develop a biomimetic polymer for cell recognition, we synthesized poly [3-O-(4'-vinylbenzyl)-D-glucose] (PVG), a polystyrene derivative with reduced glucose moiety, and studied the specific interaction of this PVG with erythrocytes, carried by a glucose transporter (GLUT-1), on a cell membrane. To clarify the specific interaction between the PVG and the erythrocytes, fluorescein isothiocyanate- (FITC) labeled polymer was used to prove and visualize the specific interaction. We found that labeled polymer strongly binds to erythrocytes, probably due to the specific interaction mediated by the presence of GLUT-1 on the cell membrane. The fluorescence intensity of PVG on erythrocytes was time and dose dependent. To verify the specific interaction between the PVG and the erythrocytes, cells were pretreated with phloretin, an inhibitor of GLUT-1, before adding the FITC-labeled PVG polymer to the cell-culture medium. This treatment suppressed the interaction of PVG with erythrocytes. A confocal laser microscopic study further confirmed this interaction. The results from this study provide evidence that a biomimetic polymer of PVG interacts with erythrocytes mediated by GLUT-1 on cell membranes.     

Catalysis of the generation of thrombin-antithrombin complex by insoluble anticoagulant polystyrene derivatives

The inhibition of thrombin by antithrombin III is known to be accelerated by heparin through the formation of complexes between the muccopolysaccharide and both proteins. In the preceding papers, we reported that polystyrene derivatives absorb thrombin and its inhibitor with a higher affinity for the protease than for the antiprotease. These complexes are responsible for the catalysis of the generation of thrombin-antithrombin complex which was observed either with purified proteins or in plasma. The protease-antiprotease complex has an affinity for the polymer surface which is higher than that of antithrombin but lower than that of thrombin. Therefore, the thrombinantithrombin complex generated on the insoluble material is desorbed by thrombin and a catalytic anticoagulant effect can be observed with these polymers.     

Sm22.6 antigen is an inhibitor to human thrombin

The physiological role of Schistosomiasis mansoni 22.6 antigen (sm22.6 Ag) and its pathogenic effect on the human host has never been reported. Recombinant sm22.6 Ag is a homogenous polymer under non-denaturing/non-reducing conditions, and an inhibitor to human thrombin. Kinetic and Western blot assays show that the recombinant protein interacts with human thrombin and inhibits proteolytic activity of the protease. Tests of whole blood revealed that coagulation time was significantly delayed (3-5 times longer) in the presence of the recombinant protein at a concentration similar to thrombin in normal blood samples. Kinetic studies revealed that the delayed coagulation time was due to the inhibition of alpha-thrombin proteolytic activity by the parasite protein in an irreversible pattern, and a reversible inhibition to gamma-thrombin. Also, Western blot analysis under non-denaturing/non-reducing conditions showed that sm22.6 Ag binds to both alpha- and gamma-thrombin. Our results strongly suggest that sm22.6 antigen plays a role in down-regulation of coagulation in humans.     

In vitro non-thrombogenicity of a thrombin-substrate-immobilized polymer surface by the inhibition of thrombin activity

Derivatives of thrombin substrate were synthesized and immobilized on a poly(acrylic acid)-grafted polyurethane film. The carboxyl terminal of the thrombin substrate peptide should be blocked for a higher inhibitory effect of the thrombin activity. Immobilization of the thrombin substrate peptide enhanced adsorption and inactivation of thrombin on the polymer film to prolong the time for fibrin network formation, and suppressed adhesion and deformation of platelets on the film. Consequently, in vitro thrombus formation on the polymer film was strongly suppressed.     

Copolymers of 2-methacryloyloxyethyl phosphorylcholine (MPC) as biomaterials

Copolymers of 2-methacryloyloxyethyl phosphorylcholine (MPC) showed good hemocompatibility as hypothesized. The hypothesis was surfaces having phosphorylcholine groups by polymerization of MPC could accumulate phospholipids from blood stream and show good blood compatibility. We designed and prepared a methacylate having a phosphorylcholine group. While it was possible to introduce them by polymer reactions, polymer reaction is not always good method to prepare the desired pure surface. This must be very important point to consider biomaterials, as we have to apply them in our body without any adverse effects.The hypothesis was confirmed by changing copolymer composition. The adsorption amount of phospholipids on the surfaces increased with increasing the MPC units in the copolymers. On the other hand, increasing MPC units in MPC copolymers decreased adsorption amount of peptides. There is limitation in blood compatibility tests in vitro due to unstable characteristics of blood itself. We evaluated them with series of blood compatibility tests, in vitro, ex vivo and in vivo, on coated PMMA beads, modified hollow fibers for hemodialysis and 2 mm small diameter blood vessels, respectively. These data suggested MPC is a promising methacrylate to develop good blood contacting devises, which may not require systemic anticoagulation. Conventional blood compatible biomaterials were not suitable to make permeable membranes. But MPC is soluble in water and we could prepare permeable membranes to various solutes by the copolymerization. Introduction of MPC copolymers on cellulose and polysulfone hollow fiber membranes gave them nonthrombogenicity but it did not give adverse effect on their permeability. These data suggested that it is possible to apply them to hemodialyzers, oxygenators and percutaneous glucose sensors to keep diabetic patients easier. MPC surfaces are good hydrogel to minimize damage on tissues by lubricating between organs and the coated devices. They do not induce denaturation of peptides, which is beneficial to keep activities of enzymes longer. And poly-MPC dissolved is applicable to stabilize several bioactive peptides in aqueous phase. So MPC polymers are useful to minimize fouling by inhibiting the adsorption of bioactive proteins. MPC has high potential to develop many varieties of new biomaterials useful in so-called biotechnology. MPC and their copolymers are commercially available from NOF (Tokyo, Japan) and Biocompatibles (UK, as PC technology).     

Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential

In order to design plasminogen activators with improved thrombolytic properties we sought to construct the bifunctional protein HLS-2 which combines both a plasminogen-activating and an anticoagulative activity. The chimeric protein comprises four elements: a derivative of thrombin inhibitor hirudin, a 6-amino acid spacer, the sequence of plasminogen-activator staphylokinase (Sak), and a 13-amino acid expression tag at the C-terminus. The gene of the fusion protein was obtained by SOE-PCR, cloned into pCANTAB5E, and expressed in E. coli BL21. HLS-2 was purified from periplasmatic extracts and characterized by Western blotting. Plasminogen-activation of HLS-2 and of Sak in equimolar mixtures with plasminogen showed near equivalence as measured by plasmin-mediated cleavage of chromogenic substrate S-2403. For catalytic amounts of plasminogen-activator, however, HLS-2 was less effective by a factor of 1.7. HLS-2 also inhibited both the amidolytic and the fibrinolytic activities of thrombin. Similar concentrations of either commercial HV1 (42 pmol/L) or HLS-2 (250 pmol/L) were required to halve the initial rate of thrombin reaction with fluorogenic substrate Tos-Gly-Pro-Arg-AMC, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein sufficiently strong to substitute anticoagulative comedication during fibrinolytic treatment. The results provide a rationale for further testing the efficacy of HLS-2 for the lysis of platelet-rich arterial blood clots and for the prevention of reocclusion after thrombolysis.     

Immobilization of an anticoagulant benzamidine derivative: effect of spacer arms and carrier hydrophobicity on thrombin binding

Prevention of blood coagulation is very often a prerequisite for successful medical devices. For that purpose, passivation of the key coagulation enzyme thrombin through the derivatization of the material's surface with an amidine-based molecule has been found to be promising. To further enhance the efficiency of this approach, thin layers of maleic anhydride copolymers offering different physico-chemical characteristics were tethered with carboxyl terminated polyethylene glycol to covalently immobilize a benzamidine-type derivative. The free carboxyl surface groups produced by the attachment of polyethylene glycol (PEG) were quantified by Ag(+) labeling and subsequent XPS detection. The film thickness as well as the carboxyl group content were found to be clearly dependent on the copolymer hydrophobicity and the nature of the PEG molecule. For the assessment of the anchorage of the thrombin to the benzamidine-derivative functionalized surfaces, the substrates were immersed in a buffered thrombin solution and the enzyme adsorption was studied using immunostaining/confocal laser scanning microscopy. Higher degrees of thrombin binding were observed for substrates configured with the hydrophilic compared to the more hydrophobic copolymer. Moreover, surface-bound spacers based on alpha,omega-heterobifunctional PEG amino acids (alphaAm,omegaAc-PEG) also enhanced the benzamidine surface density in comparison to homofunctional PEG diacids (alphaAc,omegaAc-PEG) because of a lower degree of carboxyl inactivation due to PEG 'bridging'. Altogether, the choice of copolymer coatings and the type of PEG spacers were demonstrated to enhance the efficiency of the thrombin scavenging by the covalently immobilized coagulation inhibitor.     

The ability of Sephadex to activate human complement is suppressed in specifically substituted functional Sephadex derivatives

The capacity of Sephadex and of chemically substituted Sephadex derivatives to activate human complement was examined by incubating polymer particles in normal human serum (NHS) under conditions that allow classical and/or alternative pathway activation, and by determining complement consumption or generation of C3a antigen in serum. Sephadex was found to activate complement in NHS, mainly through the alternative pathway. The complement-activating capacity of Sephadex was directly related to the surface area of polymer that could interact with serum. Substitution of hydroxyl groups of Sephadex with carboxymethyl (CM) groups suppressed the complement-activating capacity of the polymer in a dose-dependent fashion so that Sephadex bearing an average of one or more CM groups per saccharidic unit exhibited no complement-activating ability. Blocking of CM groups on CM sephadex with amide bonds did not restore a complement-activating capacity to the polymer, indicating that intact hydroxyl groups of the sugar units are required for complement activation by Sephadex. CM Sephadex was also found to adsorb C3adesArg which bound to the polymer with a calculated affinity of 1 x 10(6) l x M-1. Substitution of Sephadex with carboxymethyl and benzylamide sulphonate groups which confers to the polymer the capacity to catalyse thrombin inactivation on its surface also suppressed the complement-activating capacity of Sephadex. Sephadex derivatives that lack complement-activating properties and adsorb anaphylatoxins may provide useful models for the design of cellulosic membranes and biomaterials with blood compatible properties.     

Adsorption of plasma proteins onto anticoagulant polystyrene derivatives: a fluorescence study

Quenching of fluorescence was used to monitor adsorption of thrombin (T), antithrombin (AT) and their inactive complex (T-AT) onto three anticoagulant biomaterials made of polystyrene beads bearing the functional groups of heparin. An adsorption capacity of 0.12 mumol of T per mg of polymer allowed the formation of a monolayer of protein at the polymer surface. An affinity constant of 3 x 10(7) l.mol-1 between thrombin and polymer was estimated, whatever the polymer used. The affinity of T-AT was similar although weaker. Desorption of proteins from the polymeric interface by means of polycations (polybrene and polylysine) showed that the inactive complex T-AT is more quantitatively and easily released than thrombin.     

微小牛蜱凝血酶抑制剂的分离纯化与活性研究

从半饱吸血的微小牛蜱(Boophilusmicroplus)唾液腺中分离纯化到了一种凝血酶抑制剂,用飞行质谱测定该抑制剂的分子量为6752Da,不同于已知的从蜱类来源的丝氨酸蛋白酶抑制剂。这是世界上首次报道的从微小牛蜱唾液腺分离出来的凝血酶抑制剂。该抑制剂对凝血酶表现强烈的抑制活性,对激活的第X因子和胰蛋白酶具有微弱的抑制活性。     

A novel hemostatic delivery device for thrombin: biodegradable poly(D,L-lactide-co-glycolide) 50:50 microspheres

Topical thrombins are locally active hemostatic agents that can be used to minimize blood loss during any surgery. The aim of this study was to design and investigate a thrombin-containing biodegradable hemostyptic device with an optimized drug release profile to promote local blood clot formation. It is effective with ongoing systemic antithrombotic therapy and can be used in all types of bone-related surgery, for example, in dental surgery. Thrombin-loaded poly(D,L-lactide-co-glycolide) microspheres were synthesized by means of complex (w/o/w) emulsion evaporation method. The resulting enzyme activity of the serine-protease thrombin was verified by the specific chromogenic substrate S-2238. The thrombin release profile depended on four factors: (1) thrombin dosage, (2) polymer concentration in the o-phase, (3) phase quotient w1:0 in the primary emulsion, and (4) the addition of pore-introducing agents. A collagenous sponge containing thrombin-loaded microspheres by means of lyophilization was developed. The impact of several production factors of the (w1/o/w2) solvent evaporation method to optimize thrombin encapsulation, morphology of the spheres, and desired drug release profile have been investigated. The in vitro thrombin release was dependent on the polymer-to-oil phase ratio, the polymer concentration, and the type of solvent and polymer. The porosity of the spheres and release rate of the active agent were enhanced by increasing the inner aqueous w1 phase. With this study, a new biodegradable hemostyptic device could be verified and established for a potentially safe and locally controlled thrombin release to manage postsurgical hemorrhage in patients undergoing anticoagulant therapy.     

Controlled preparation of thin fibrin films immobilized at solid surfaces

A technique for coating surfaces with attached fibrin structures without the formation of fibrin gel in bulk solution was developed. It is based on the catalytic effect of the surface-bound thrombin on fibrinogen stabilized with inhibitor which inhibits thrombin in solution but not the thrombin on the surface. Such an inhibitor is antithrombin, the effect of which may be enhanced with heparin. Fibrinogen is first adsorbed on the substrate surface and then incubated with thrombin. The unbound thrombin is washed out and the surface is incubated with fibrinogen solution containing antithrombin III and heparin. A fibrin gel forms at the surface by the action of surface-bound thrombin on ambient fibrinogen solution; however, the gel formation in bulk solution catalyzed by thrombin partially released from the surface is suppressed. By utilizing antithrombin-independent inhibitors or repeating thrombin binding and incubation with fibrinogen solution, the amount of surface-attached fibrin gel can be controlled. The formation of immobilized fibrin networks was observed using surface plasmon resonance and turbidity measurements and morphology was observed by TEM, SEM, and AFM. Using this technique, a porous scaffold made of polylactide fibers was coated with fibrin without filling the space between fibers with a bulk fibrin gel. The technique makes it possible to coat the inner surface of porous scaffolds with surface-attached fibrin gel while preserving free volume for cell migration into the pores.     

Evaluation of coagulation tests in mouse plasma

The coagulation variables thrombin time (TT), activated partial thrombin time (APTT) and prothrombin time (PT), were investigated in mouse plasma. TT and APTT clotting times were determined using a KC10 coagulation analyser and test kits Dia Thrombin (bovine thrombin diluted to a concentration of 2.5 U/l) and Dia Celin-L (rabbit brain cephaloplastin, activated with complexed Kaolin), respectively. PT was determined with IL Test™ Hepatocomplex (rabbit brain thromboplastin and calcium ions) using an ACL 200. Furthermore, the test procedures were also used to assess the anticoagulant status of mice treated orally with Melagatran, a thrombin inhibitor. The results showed that the kits could be used successfully on mouse plasma to measure the effect of a thrombin inhibitor on haemostasis.     

Affinity of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: I. In vitro adsorption studies

In previous papers, we described insoluble polystyrene derivatives which exhibit a heparin-like antithrombic activity in plasma. In order to ascertain the heparin-like mechanism of this activity we have studied the interactions of thrombin and antithrombin III with two polymers of this series: sulphonated polystyrene and sulphonate-glutamic acid sulphonamide polystyrene. The adsorption was measured using purified enzyme and enzyme inhibitor and polymer beads whose average diameter was about 25 μm. The maxima of adsorption approximately correspond to a monolayer of protein. The results are discussed with respect to the most common isotherms used in chemisorption and the affinities of the enzyme and its inhibitor for both materials are evaluated: k_T- 10⁷(M/I)⁻¹, k_AT- 3.10⁵(M/I)⁻¹.     

Thrombin impairs alveolar fluid clearance by promoting endocytosis of Na+,K+-ATPase

Coagulation is an emerging area of interest in the pathogenesis and treatment of acute lung injury. Concentrations of the edemagenic coagulation protease thrombin are elevated in plasma and lavage fluids from afflicted patients. We explored the impact of thrombin on the formation and resolution of alveolar edema. Intravascularly applied thrombin inhibited active transepithelial 22Na transport in intact rabbit lungs, suppressing alveolar fluid clearance. Epithelial permeability was unaffected, whereas endothelial permeability was increased. In A549 human lung epithelial cells and in mouse primary alveolar type II cells, thrombin blocked ouabain-sensitive Na+,K+-ATPase-mediated 86Rb+ uptake, without altering amiloride-sensitive sodium currents. Furthermore, thrombin downregulated cell-surface expression of Na+,K+-ATPase, but not ENaC alpha and beta subunits. The endocytosis inhibitor phalloidin oleate blocked all thrombin-induced effects on sodium transport activity. Similarly, diphenyleneiodonium chloride, an inhibitor of reactive oxygen radical production, as well as a protein kinase C-zeta inhibitor, prevented these thrombin-induced effects. Thus, thrombin signaling via reactive oxygen species and protein kinase C-zeta promotes Na+,K+-ATPase endocytosis, resulting in loss of function. We propose here a dual role for thrombin in mediating disturbances to fluid balance in the lung: thrombin concomitantly provokes edema formation by increasing endothelial permeability, and inhibits alveolar edema resolution by blocking Na+,K+-ATPase function.     

Role of the extracellular signal-regulated kinase 1/2 signaling pathway in the process of thrombin-promoting airway remodeling in ovalbumin-allergic rats

Abstract

Context: Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event.

Objective: In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats.

Materials and methods: A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-β1 and IL-6 was detected by ELISA.

Results: The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment.

Conclusion: These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.