Anticoagulant activity of dextran derivatives. Part II: Mechanism of thrombin inactivation

The mechanism of anticoagulant activity of dextrans substituted with carboxylic and benzylamide sulphonate groups is studied by various coagulation tests. These derivatives exhibit a heparin-like antithrombic activity which requires the presence of antithrombin III; however they are less effective than heparin on a weight basis. They also exert a direct antithrombic activity by an antithrombic III independent pathway; but this action is negligible compared to the thrombin inhibition observed in the presence of antithrombin III. Dextran derivatives have been prepared with antithrombic properties similar to those of heparin.     

Anticomplementary activity of dextran derivatives

Substitution with carboxylic and benzylamine sulphonated groups conferred on dextran both antithrombic activity and the capacity to inhibit formation of the amplification C3 convertase of complement. In dextrans substituted with carboxylic groups (greater than 40%), a high content of sulphonate (greater than 10%) resulted in both anticomplementary and antithrombic properties whereas a lower content of sulphonate resulted in high anticomplementary but weak antithrombic activity. The anticomplementary activity of highly substituted dextrans was similar to that of heparin, although anticoagulant activity was much lower than in heparin, confirming independent structural requirements for both activities in the heparin molecule.     

Anticoagulant activity of dextran derivatives. Part I: Synthesis and characterization

Substituted dextrans bearing carboxymethyl and benzylsulphonate groups have been prepared. These materials exhibit an antithrombic activity correlated with the ratio of each substituent. The highest activity is obtained when the dextran derivative contains more than 50% of carboxylic acid groups and simultaneously about 15% of benzylsulphonate functions.     

Anticoagulant activity of original synthetic peptide derivatives

Original synthetic peptide derivatives exhibit anticoagulant activity in vitro and in vivo. They delayed fibrin clot formation from human blood plasma in tests for the intrinsic coagulation pathway (activated partial thromboplastin time) and final stage of plasma coagulation (thrombin time) and inhibited amidolytic activity of thrombin. We determined the minimum effective dose of the most active compound providing a 2-fold lengthening of blood clotting time (activated partial thromboplastin time test and thrombin time test), which persisted for 2–3 h. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 1, pp. 57–60, January, 2008     

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.     

Inhibition of thrombin and factor Xa by <Emphasis Type="Italic">Fucus evanescens</Emphasis> fucoidan and its modified analogs

Specimens of fucoidan extracted from Fucus evanescens were purified from protein and polyphenols, deacetylated and depolymerized by fucoidanase for evaluation of their biological activity. Deacetylation did not modify the capacity of fucoidan to inhibit thrombin and factor Xa, while purification from protein and polyphenols reduced this capacity. Depolymerization of fucoidan increased its capacity to inhibit thrombin mainly through heparin cofactor II. All the studied specimens formed complexes with protamine sulfate. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 9, pp. 304–309, September, 2008     

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.     

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.     

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.     

Study of neurotrophic activity of thrombin on the model of regenerating mouse nerve

Experiments demonstrated a dose-dependent facilitating effect of thrombin and peptide thrombin receptor agonist PAR1 (TRAP6) on regeneration of mouse peripheral nerve after its crushing. The maximum neurotrophic effect was observed at low concentrations of thrombin (10 nM) and TRAP6 (10 µM).__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 8–10, January, 2005     

Correction effect of ATP on platelet aggregation and blood coagulation <Emphasis Type="Italic">In Vitro</Emphasis> and in the presence of circulating thrombin in rats

ATP added to plasma samples in concentrations of 5×10⁻³−5×10⁻⁵ M in vitro decreased ADP-induced platelet aggregation. Platelet aggregation stimulated with thrombin under similar experimental in vitro conditions significantly decreased in the presence of 5×10⁻⁶ M ATP and tended to decrease under the influence of ATP in concentrations of 5×10⁻³ and 5×10⁻⁷−5×10⁻⁹ M ATP. When endogenous thrombin in the circulation was stimulated by intravenous infusion of tissue thromboplastin, pretreatment with ATP (4 intramuscular injections, 0.75 mg/kg) produced a correction effect and normalized disturbed anticoagulant activity and platelet aggregation. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 364–366, April, 2007     

The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin

The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to ±80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin.     

The mechanism of anticoagulant activity of a novel heparinoid sulfated glucoside-bearing polymer

In our previous study on glycoside polymer, it was discovered that sulfated poly(glucosyl-oxyethyl methacrylate) [poly(GEMA)-sulfate], which bears sulfated D-(+)-glucose, exhibited anticoagulant activity. The anticoagulant activity of poly(GEMA)-sulfate in solution was prolonged by increasing the dose or degree of sulfation of the polymer. In this study, to recognize the mechanism of anticoagulant activity of poly(GEMA)-sulfate, we estimated the binding capacity of antithrombin III to thrombin and some in vitro clotting tests in the presence of poly(GEMA)-sulfate. These results revealed that poly(GEMA)-sulfate had an anticoagulant mechanism which differed to that of heparin. We concluded that the anticoagulant activity of poly(GEMA)-sulfate is responsible for inhibiting fibrin network formation by the insoluble ion complex between fibrinogen and poly(GEMA)-sulfate.     

Effect of a derivatized dextran on human osteoblast growth and phenotype expression

Water soluble derivatized dextran named E9 with a molecular weight of 45 000 g 1^-1 containing 58% methyl carboxylic acid unit, 19% benzylamide unit, and 26% sulfonate with a specific anticoagulant activity of 0.29 IU mg^-1 was studied for its effects on human osteoblast growth and phenotype expression for short-term treatment. At concentrations between 1 ng ml^-1 and 1 μg ml^-1 E9 has no effect on DNA synthesis whereas at higher concentrations DNA synthesis is inhibited in a dose related fashion (87% for 400 μg ml^-1). For concentrations which do not modify osteoblast growth, E9 promotes alkaline phosphatase activity, type I collagen and osteocalcin synthesis with a maximum effect for 0.1-1 μg ml^-1. It has a synergistic effect with hPTH increasing AMPc. Moreover, osteonectin synthesis was enhanced in a dose-dependent manner between 0.1 and 5 μg ml^-1. These results seem to indicate that E9 is able to stimulate human osteoblast phenotype expression and could be useful in clinical applications.     

Preparation,characterization and in vitro anticoagulant activity of corn stover xylan sulfates

A new anticoagulant agent was prepared by introducing sulfate groups into corn stover xylan through homogeneous reactions. Three organic solvents, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and formamide (FA), were adopted as reaction media, with the assistance of LiCl. Structural characterization by FT-IR and ¹³CNMR showed that xylan sulfate (XS) could be successfully synthesized with SO₃?Pyridine (SO₃?Py) complexes sulfation reagent in the three media. The effect of sulfation temperature, sulfation time, media type and molar ratio of ?SO₃/?OH on the degree of substitution (DS) and degree of the polymerization (DP) were studied. DMF/LiCl were more effective than DMSO/LiCl and FA/LiCl in preparation of xylan sulfate with high DS. The optimal conditions for sulfation were obtained when SO₃?Py complex was added to DMF/LiCl with ?SO₃/?OH ratio of 1.5:1 and maintained at 50 °C for 3 h. Degree of polymerization of xylan was decreased during the sulfation process and DMF/LiCl offered the least xylan degradation as compared with DMSO/LiCl or FA/LiCl. Anticoagulant activities of the resultant xylan sulfates with different DS were evaluated by using activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). Results indicated that the introducing of sulfate groups into xylan did endow the polysaccharides with anticoagulant activity. The APTT and TT of XS with DS of 1.20 reached 141 and 45.3 s at a dosage of 20 μg/mL, while the APTT and TT values for the blank sample were only 35.5 and 15.6 s. Furthermore, coagulation time was prolonged with the increase of DS and the concentration of XS. Our findings provide new insights into the value-added utilization of agricultural biomass.     

Radiation cross-linked collagen/dextran dermal scaffolds: effects of dextran on cross-linking and degradation

Ionizing radiation effectively cross-links collagen into network with enhanced anti-degradability and biocompatibility, while radiation-cross-linked collagen scaffold lacks flexibility, satisfactory surface appearance, and performs poor in cell penetration and ingrowth. To make the radiation-cross-linked collagen scaffold to serve as an ideal artificial dermis, dextran was incorporated into collagen. Scaffolds with the collagen/dextran (Col/Dex) ratios of 10/0, 7/3, and 5/5 were fabricated via ⁶⁰Co γ-irradiation cross-linking, followed by lyophilization. The morphology, microstructure, physicochemical, and biological properties were investigated. Compared with pure collagen, scaffolds with dextran demonstrated more porous appearance, enhanced hydrophilicity while the cross-linking density was lower with the consequence of larger pore size, higher water uptake, as well as reduced stiffness. Accelerated degradation was observed when dextran was incorporated in both the in vitro and in vivo assays, which led to earlier integration with cell and host tissue. The effect of dextran on degradation was ascribed to the decreased cross-linking density, looser microstructure, more porous and hydrophilic surface. Considering the better appearance, softness, moderate degradation rate due to controllable cross-linking degree and good biocompatibility as well, radiation-cross-linked collagen/dextran scaffolds are expected to serve as promising artificial dermal substitutes.     

Cultivation of human immunodeficiency virus from whole blood: Effect of anticoagulant and inoculum size on virus growth

Human immunodeficiency virus (HIV) was cultivated directly from whole blood treated with anticoagulant by cocultivation with phytohaemagglutinin-stimulated cord blood lymphocytes. When heparin was used as the anticoagulant, isolation rates were low (10% to 56%, depending on the patient group); but when EDTA was used, isolation rates were much higher (50% to 100%). Culture of whole blood gave results identical to those of culture of separated peripheral mononuclear cells, and in some cases virus could be isolated from as little as 10 μl of unseparated EDTA anticoagulated blood.