Excited-state dynamics of ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate.

The photophysics and excited-state dynamics of ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate (EAADCy) in solvents of varying polarity and viscosity have been studied using femtosecond transient absorption spectroscopy. Global analysis of the time-resolved spectra revealed three processes occurring in an optically excited molecule. The sequence of reactions begins with a transition from an initially excited Franck-Condon state to the nonrelaxed intramolecular charge transfer (ICT(NR)) state which is associated with a partial electron transfer. This process is followed by an additional relaxation to a more relaxed intramolecular charge transfer (ICT(R)) state with stronger charge transfer character and flattened geometry. The lifetime of the flattened charge transfer form (ICT(R)) shortens from 200 to 300 ps in medium polar solvents down to 10 ps in strongly polar solvents. On the other hand, increase of viscosity by 1 order of magnitude leads to deceleration of processes involving twist of the donor and acceptor moieties by a factor of approximately 2.5. Observation of long-lived fluorescence of EAADCy in medium polar solvent suggests that charge transfer is possible only from a hot Franck-Condon state, but not from a relaxed locally excited state which exhibits short-wavelength fluorescence on a nanosecond time scale.

Influence of prototropic reactions on the absorption and fluorescence spectra of methyl p-dimethylaminobenzoate and its two ortho derivatives.

The influence of prototropic reactions on the spectral characteristics of methyl p-dimethylaminobenzoate (I) and its o-methoxy (II) and o-hydroxy (III) derivatives has been studied using steady-state spectroscopic technique and quantum-chemical calculations. This study concerns the solvent-induced shift of the absorption, locally excited (LE) and intramolecular charge transfer (ICT) fluorescence bands in the neat tetrahydrofuran (THF) and its hydrochloric acid solutions at different HCl concentrations. On the basis of the experimental results and quantum-chemical calculations, it was shown that in a hydrochloric acid solution the studied molecules exist as a mixture of neutral, mono-, and dicationic forms. Additionally, the results of spectroscopic measurements were used to calculate, according to the Benesi-Hildebrand method, the equilibrium constants of protopropic reactions in the ground, S(0), and excited, S(1), states. Our findings predestine molecules I and II to be used as acid fluorescence probes in a region of 0-2.5 M of [H(+)] concentrations.

Mechanism of factor IXa inhibition by antithrombin in the presence of unfractionated and low molecular weight heparins and fucoidan.

Heparin exerts its anticoagulant activity by catalysing the inhibition of coagulation proteases by antithrombin (AT). Its main target is thrombin but it also catalyses the inhibition of the other serine-proteases of the coagulation cascade, such as factor IXa (fIXa). The aim of this study was to compare the catalysis of inhibition of blood fIXa by antithrombin in the presence of several sulfated polysaccharides with anticoagulant activity, i.e. heparin, three widely used in therapeutics low molecular weight heparins (LMWH) and fucoidan. Plots of the second-order rate constants of the fIXa-antithrombin reaction vs. the concentration of added heparin and LMWH are bell-shaped and fit the kinetic model established for thrombin-antithrombin reaction by Jordan R., Beeler D., Rosenberg R. (1979) J. Biol. Chem., 254, 2902-2913. In the ascending branch, the catalyst (C) binds quickly to the inhibitor (I) to form a catalyst-inhibitor (CI) complex which is more reactive towards the enzyme (E) than the free inhibitor, leading to the formation of an inactive enzyme-inhibitor complex (EI) and the release of free catalyst, in a rate-limiting second step. After a maximum corresponding to an optimal catalyst concentration, the decrease in the reaction rate was in keeping with the formation of a catalyst-enzyme (CE) complex, whose inactivation by the CI complex was slower than that of the free enzyme. Maximum second-order rate constants for the inhibition of fIXa by AT were 105, 6.8, 12.24 and 22 microM-1 min-1 with heparin, Enoxaparin, Fraxiparin and Fragmin, respectively, leading to 3500-, 225-, 405- and 728-fold increases in the inhibition rate in the absence of polysaccharide, respectively. Fucoidan yielded 23-fold increase in the fIXa-antithrombin interaction rate. The kinetic profiles obtained with this polysaccharide exhibited ascending branch which correlated well with the kinetic model based on the formation of binary complexes (CI or CE). Fucoidan was covalently conjugated with a fluorescent probe (DTAF) and used in conjunction with fluorescence anisotropy to follow its binding to antithrombin, heparin cofactor II (HCII), thrombin and fIXa. The binding of fucoidan to these proteins occurred with low affinities when compared to heparin and LMWH. Fucoidan had higher affinity for the inhibitor HCII compared to antithrombin and enzymes. These data suggest that binding of heparins and fucoidan to the inhibitor (CI) is required for the polysaccharide-dependent enhancement in the rate of neutralization of the enzyme by the 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.

Interactions between human plasma proteins and heparin-poly(methyl methacrylate) copolymer.

A solid Heparin-PMMA copolymer has been synthetized by a radical polymerization of methyl methacrylate from oxidative reaction initiated by Ce4+ ions in the presence of heparin. Covalently linked heparin was 10% of copolymer weight. The antithrombin activity of the copolymer corresponded to 1% of grafted heparin. PMMA sequence of the copolymer played the leading role in fibrinogen, immunoglobulins, transferrin and albumin adsorption. These proteins adsorbed on the copolymer, showed different competitive desorption pattern in the presence of whole plasma: fibrinogen presented the highest degree of affinity for the copolymer. The heparin part of the copolymer was responsible for antithrombin III adsorption and for decrease of factor V activity. Active antithrombin III was eluted. An inactivation of factor V in plasma was observed using high concentrations of soluble heparin. This result suggested that copolymer heparin chains, even devoid of antithrombin activity were involved in this inactivation. With Heparin-PMMA copolymer, plasma clotting pro-enzymes behaved differently than on heparin-sepharose copolymer:disappearance of factor XI activity, decrease in prekallikrein activity and activation of factor IX were observed. PMMA sequences were responsible for factor IX activation.

Insoluble DNA-like phosphorylated polystyrene: specific interactions with anti-DNA antibodies from systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disease. Antibodies directed mainly against DNA and/or phospholipids are present in the serum of SLE patients. Therefore phosphorylated polystyrene derivatives acting as DNA-like polymers should be able to interact with the SLE anti-DNA antibodies. Such functional polymers were synthesized and subsequently their interactions with the anti-DNA antibodies studied. Adsorption experiments performed with both anti-DNA antibodies and normal immunoglobulins showed high affinity constants of the phosphorylated polymer for anti-DNA antibodies (4 x 10(9) M-1) whereas for normal IgG the affinity was low (2 x 10(5) M-1). Moreover, the interaction was specific involving the idiotypic moiety of the anti-DNA antibodies and an array of phosphoester groups at the surface of this biomaterial.

Randomness and biospecificity: random copolymers are capable of biospecific molecular recognition in living systems.

Biospecific molecular recognition in living systems is known to be based on the lock and key principle as proposed by Emil Fischer. Based on this concept, biospecific polymers have been produced synthetically by attaching biospecific 'keys' to the polymer chain. We postulate that biospecificity can be achieved by alternative means, namely random substitution of a preformed polymer with suitable chemical groups or random copolymerization of suitable functional monomers. Such polymers, we suggest, will contain arrangements of the chemical functions which mimic natural biospecific sites and the probability of occurrence of such arrangements will depend on the average composition of the polymer. In support of this principle, we have developed several functional random copolymer systems which possess a variety of biological properties depending on the type of chemical function. Examples are: polymers possessing anticoagulant properties similar to those of heparin; polymers which interact specifically with components of the immune system; and polymers which, in contact with cells, affect their growth and metabolism. In the case of statistical copolymers possessing 'DNA-like' properties obtained by phosphorylation of hydroxylated polystyrene derivatives, Monte Carlo simulations were used to determine the distribution of phosphodiester (PDE) groups along the chains and to compute the probabilities of occurrence of particular arrangements of PDE found in the 'DNA-like' sites. The results showed that these sites are made up of PDE groups separated by distances that closely match those between the same groups along a generatrix of the DNA double-helix cylinder. These findings offer the prospect of manufacturing polymeric biomaterials endowed with biomimetic character. Moreover, they provide the basis for a hypothesis regarding the appearance of biospecificity at the origin of life, suggesting that biospecific structures may have evolved by natural selection from purely random copolymers. It is likely therefore that biospecificity is a continuous function of randomness, arising from purely statistical distributions of reactivity and evolving into precisely defined structures such as those involved in ligand-receptor interactions.

Heparin-like tubings. III. Kinetics and mechanism of thrombin, antithrombin III and thrombin-antithrombin complex adsorption under controlled-flow conditions.

In previous papers, we described treated tubular materials which exhibit an heparin-like antithrombic activity under dynamic conditions. In order to ascertain the heparin-like mechanism of this activity, we have studied the interactions of thrombin, antithrombin III and thrombin-antithrombin III complex with the inner face of these treated tubings under controlled-flow conditions. Moreover, the kinetics of the adsorption of thrombin were studied at different flow rates to establish the rate-determining step.

Heparin-like tubings. II. Mechanism of the thrombin-antithrombin III reaction at the surface.

In a previous paper we described the surface treatment of tubings made of polystyrene grafted by irradiation onto polyethylene tubular materials. As a result, polystyrene moieties of their inner face were substituted by sulphonate and aspartic acid sulphamide groups. In order to study the mechanism of the thrombin-antithrombin III reaction occurring at the modified surface and to determine the kinetics of the reaction, step by step experiments were set up involving either the protease adsorbed at the surface reacting with the antiprotease circulating in the solution or the opposite. These procedures allowed us to demonstrate the heparin-like catalytic activity of the tubings.

Heparin-like tubings. I. Preparation, characterization and biological in vitro activity assessment.

In order to prepare tubular materials which could be used in blood-circulating medical devices, polystyrene was grafted by irradiation on to polyethylene tubings. A chemical surface treatment was used which resulted in the functionalization of the inner face of the tubing. This procedure is described and the chemical assessment of the constitution of the functionalized polymer has been completed. Tubing, the inner face of which is made of polyethylene-polystyrene copolymer in which polystyrene moieties were substituted with sulphonate and aspartic acid sulphamid groups, was tested for antithrombic properties in a circulating device under controlled transport conditions and by use of purified proteins.

Biospecific interactions of vitamin K-dependent factors with phospholipid-like polystyrene derivatives. Part II: factor IX.

We previously demonstrated that phosphorylated polystyrene derivatives exhibit phospholipid-like behaviour and therefore are able to interact with factor II, one of the vitamin K-dependent coagulation factors. Under the same conditions as for factor II, we examined the interactions of factor IX with phosphorylated resins of various compositions in phosphate groups: these studies were carried out with or without albumin precoating of the polymers and either in the presence or absence of calcium ions. Adsorption experiments show that, in the absence of calcium ions, only one class of adsorption sites of factor IX can be evidenced with the interactions taking place through the formation of binary complexes, whereas in the presence of calcium ions, the affinity of factor IX for phosphorylated resins becomes very high and two types of adsorption sites have been evidenced with biospecific ternary complexes being formed. The domains of predominance of these complexes were determined. Moreover, the only functional groups borne by the phosphorylated polystyrene resins involved in factor IX-polymer interactions are phosphodiester groups. Comparison between factor II and factor IX adsorption onto the same polymers leads to the conclusion that the observed differences probably reflect the differences in the Gla domains of the vitamin K-dependent factors. Finally, this study demonstrates that phosphorylated polystyrene derivatives can be used as stationary phases for purification of factor IX by highly specific liquid biochromatography.

Preparation of an asymmetric semipermeable membrane with anticoagulant activity.

In this paper we report the preparation of a new asymmetric semipermeable styrene-isoprene-styrene block-copolymer membrane. Its modification by addition of gaseous N-chlorosulphonylisocyanate alters neither its permselectivity nor its water permeability rate. This modified membrane possesses an antithrombic activity which depends on antithrombin III. The actual active surface in contact with proteins is very large because the whole macroporous underlayer is modified and accessible to the proteins.

Biospecific interactions of Vitamin K-dependent factors with phospholipid-like polystyrene derivatives. Part I: Factor II.

Phosphorylated polystyrene derivatives with different compositions in phosphate groups were shown to be either recognized as phospholipidic or as DNA-like surfaces by antibodies from Systemic Lupus Erythematosus patients. In order to check whether these polymers were able to interact with Vitamin K-dependent coagulation factors, phosphorylated resins of various compositions in phosphate groups were assessed with regard to their interactions with Factor II, one of the Vitamin K-dependent factors. These studies were performed either in the presence or the absence of calcium ions, and with or without albumin precoating of the polymers. The results show that the affinity of the protein for the polymer is increased in the presence of calcium ions and depends on the composition of the polymer. The protein-polymer interactions involve the formation of binary or ternary complexes and the domains of predominance of these complexes were determined as a function of the calcium ion concentration in the assay. This allowed us to propose optimal conditions for Factor II purification by highly specific liquid chromatography using phosphorylated polystyrene resins of given compositions as stationary phases.

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.

Contributions of negatively charged chemical groups to the surface-dependent activation of human plasma by soluble dextran derivatives.

Negatively charged surfaces are known to promote contact activation. The mechanism responsible for increasing affinity for surfaces is not yet quite understood, although the presence of negative charge densities is thought to be a prerequisite. With the availability of soluble dextran derivatives, varyingly substituted with charged methylcarboxylate, methylbenzylamide sulphonate and uncharged methylbenzylamide residues, we were able to discriminate between the contributions of these chemical moieties to contact activation, thus suggesting that the stimulating properties of synthetic negatively charged surfaces should also be described in terms of specific interactions instead of global negative charge density. This could be effected by quantifying the activating capacities as a function of the chemical group composition. A direct correlation linking activating capacities to anticoagulant properties has been observed.

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 micron. 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: kT congruent to 10(7) (M/I)-1, kAT congruent to 3.10(5) (M/I)-1.

Anticoagulant activity of amino acid modified polystyrene resins: influence of the carboxylic acid function.

In previous papers, we have described the preparation and heparin-like properties of insoluble modified polystyrene resins. We now report results obtained with amino acid sulphamide resins that are virtually devoid of sulphonate groups and with resins bearing both sulphonate groups as well as amino acid sulphamides with spacers of various lengths. The absence of sulphonate groups does not affect the anticoagulant activity of the former type of resin. The biologic activity of the latter type of resin is dependent upon the length of the spacer between the amino and carboxylic acid functions. Maximal anticoagulant potency is attained with a spacer consisting of three methylene groups. Biologic activity is reduced with spacers that are less than or greater than this critical size.

Adsorption of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: II. Competition with the other plasma proteins.

In the preceding paper, we described results concerning the adsorption of purified thrombin and antithrombin III on two insoluble anticoagulant polystyrene derivatives. We now report similar results obtained in a plasma system. In each case, the purified protein was mixed with fresh platelet poor plasma in order to maintain the same concentrations of all the other plasma proteins. The thrombin molecule was modified by alkyl phosphorylation of the active serine site prior to mixing with plasma. The adsorption of antithrombin was found to be reduced 8 to 9 times when the protein solution was substituted by diluted plasma. In contrast, the thrombin adsorption only depends on the substituents bound on the polymeric chain. These results are supported by those of the study of the competition between purified antithrombin and albumin.

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 thrombin-antithrombin complex generated on the insoluble material is desorbed by thrombin and a catalytic anticoagulant effect can be observed with these polymers.

Heparin-like activity of insoluble sulphonated polystyrene resins. Part III: Binding of dicarboxylic amino acids.

It has been demonstrated previously that polystyrene sulphonate possesses anticoagulant properties and that the binding of some amino acids could enhance the heparin-like properties of such resins. These properties depend on the surface density of the active groups, the nature and binding of the group and on the net change borne by the polymer. In this paper, we describe the preparation of copolystyrene (sulphonate-dicarboxylic amino acid sulphamide) resins. By measuring their antithrombotic-surface-activity, we demonstrate that the activity developed by each carboxyl group is at least roughly the same as the activity of one sulphonate group, except in the case of aspartic acid sulphamide resin for which a cooperative effect is shown. The anticoagulant properties of resins bearing phosphonate or monocarboxylic amino acid sulphamides are also examined.