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.

Human adult tonsil xenotransplantation into SCID mice for studying human immune responses and B cell lymphomagenesis.

OBJECTIVE: To generate a human-mouse xenochimeric model where human cells remain clustered in the animal to optimize their interactions and recovery. MATERIALS AND METHODS: Severe combined immune deficient mice (SCID) were xenografted subcutaneously with human adult tonsil pieces (hu-ton-SCID mice). Such animals were: (a) compared with those receiving tonsil cells in suspension, and (b) immunized with de novo and recall antigens. RESULTS: Human tonsil pieces survived a long period of time in SCID mice, while polyclonal human T- and B-lymphocytes persisted in close vicinity within the implantation area; however, little or no graft-versus-host disease was detectable. Not surprisingly, local development of lymphoproliferative disease was often observed in animals receiving lymphoid implants from donors previously infected by the Epstein-Barr virus. One month after surgery, higher serum levels of human IgG were found in SCID mice transplanted with tonsil pieces (2x10(7) cells/animal) than in animals injected with 5x10(7) tonsil cells in suspension (1.9 vs. 0.3 mg/mL, p < 0.002). Importantly, the production of human IgG in hu-ton-SCID mice remained polyclonal for at least 6 months and was linked to the presence of cells within the implants. Immunization of hu-ton-SCID mice with hepatitis B core, a de novo antigen, did not produce a significant IgG immune response; however immunization with tetanus toxoid (TT), a thymus-dependent recall antigen, yielded high (> 700-fold increase in anti-TT IgG levels) and long-lasting (> 6 months) secondary immune responses. CONCLUSION: The hu-ton-SCID mouse xenochimeric model described in this report may improve our understanding of human lymphoid cell interactions, secondary immune responses, and lymphomagenesis.

Venous antithrombotic and anticoagulant activities of a fucoïdan fraction.

Fucoïdans catalyse thrombin inhibition by antithrombin (AT) and heparin cofactor II (HCII); their affinity for each serpin varies according to the seaweed species from which they are extracted, as well as their chemical composition and molecular weight. We extracted a homogeneous fucoïdan fraction from Ascophyllum nodosum, a brown seaweed, and tested its anticoagulant and antithrombotic activities. At a fucoïdan concentration of 3.75 micrograms/ml, thrombin inhibition mediated by AT showed an apparent second-order rate constant (kapp) of 2 x 10(8) M-1 min-1, compared to 1.5 x 10(6) M-1 min-1 for the uncatalyzed reaction. The kapp value of thrombin inhibition via HCII was 1.17 x 10(9) M-1 min-1 at a fucoïdan concentration of 50 micrograms/ml, compared to 1.72 x 10(5) M-1 min-1 for the uncatalyzed reaction. In a Wessler model of venous thrombosis, the fucoïdan fraction, injected intravenously to rabbits 10 min before thrombosis induction, exhibited antithrombotic activity: 1.8 mg/kg was the dose which inhibited F Xa-induced thrombus formation by 80% (ED80), compared to a heparin ED80 of 0.1 mg/kg. At this ED80 the antithrombotic effect of the fucoïdan persisted longer than that of heparin (30 min versus 15 min). The thrombin clotting time (TCT) was significantly prolonged (73 s versus control 29 s, compared to 53 s with heparin) 10 min after a fucoïdan bolus infusion giving a plasma fucoïdan concentration of 14.6 +/- 2.7 micrograms/ml. The bleeding time was slightly increased after fucoïdan infusion at the ED80. Fucoïdan extracted from marine flora thus shows promise as an antithrombotic drug.

Antithrombotic and anticoagulant activities of a low molecular weight fucoidan by the subcutaneous route.

Fucoidans (high-molecular-weight sulfated polysaccharides extracted from brown seaweeds) have anticoagulant and antithrombotic effects. They inhibit thrombin by catalyzing both serpins (antithrombin and heparin cofactor II) according to their chemical structures and origins. In this study, a low-molecular-weight (LMW) fucoidan of 8 kDa was obtained by chemical degradation of a high-molecular-weight fraction. The antithrombotic and anticoagulant activities of this new compound were compared to those of a low-molecular-weight heparin (LMWH), dalteparin, following subcutaneous administration to rabbits. This LMW fucoidan exhibited dose-related venous antithrombotic activity, with an ED80 of about 20 mg/kg, 2 h after a single subcutaneous injection. Its activity was comparable to that of dalteparin (close to 200 anti-Xa IU/kg) and was maximal 30 min after a single subcutaneous injection. The activity remained stable (about 70%) from 1 to 4 h after injection, but disappeared by 8 h. The antithrombotic activity was not associated with either a prolongation of the thrombin clotting time (TCT) or an increase in anti-Xa activity, contrary to dalteparin. A slight prolongation of APTT occurred with both compounds. This venous antithrombotic activity was associated with a decrease in ex vivo thrombin generation and with a significant increase in the lag phase in a thrombin generation test. LMW fucoidan thus has potent antithrombotic activity and a potentially weaker haemorrhagic effect (i.e. a smaller effect on coagulation tests and a smaller prolongation of the bleeding time) than dalteparin.

Human peripheral blood leukocyte engraftment into SCID mice: critical role of CD4(+) T cells.

We examined the influence of donor T lymphocytes on human peripheral blood leukocytes (PBL) engraftment into severe combined immune deficient (SCID) mice. Mice were injected with unfractionated or subset-depleted human PBL, and treated at various times with OKT3, a cytotoxic monoclonal antibody against human CD3(+) T lymphocytes. PBL engraftment, high levels of human Ig, and high incidence of lymphoproliferative disease (lpd) were found in mice transplanted with unfractionated PBL and CD8- or CD14-depleted PBL, and in mice treated with OKT3 at distance from PBL transfer. Animals xenografted with CD3- or CD4-depleted PBL, or treated at transplantation time with OKT3, had very low levels of human Ig and did not develop lpd. PBL engraftment was minimal or absent in these animals, as determined by immunohistochemistry, dot-blot, and RT-PCR analyses. These results demonstrate that the presence of donor CD4(+) T lymphocytes at transplantation time is necessary for observing human PBL engraftment into SCID mice, an essential condition for human Ig production and lpd development.

Comparative anticoagulant activity and influence on thrombin generation of dextran derivatives and of a fucoidan fraction.

CMDBS compounds are synthetic dextran derivatives with a random distribution of glucosyl units substituted with carboxymethyl, benzylamide, sulfonate, and sulfate groups. Fucoidans are sulfated polysaccharides extracted from brown seaweeds. CMDBS and fucoidans exhibit anticoagulant activity which depends on their chemical composition and molecular weight. Tested with purified proteins, these compounds catalyse thrombin (EC 3.4.21.5) inhibition mainly via heparin cofactor II (HCII). We investigated the mechanism involved in the anticoagulant activity of these polysaccharides relative to that of heparin. Three CMDBS with different chemical compositions were studied to evaluate the effect of sulfate and sulfonate groups on the anticoagulant activity. The fucoidan fraction was extracted from the brown seaweed Ascophylum nodosum. The clotting assays (activated partial thromboplastin time, thrombin time, prothrombin time) were significantly prolonged in the presence of CMDBS and fucoidan, which were less active than heparin. To investigate the action mechanism of these polysaccharides, thrombin generation tests (TGT) were performed on human plasma in the presence of several CMDBS and a fucoidan fraction. The results showed an inhibition of thrombin generation in contact-activated plasma in the presence of both polysaccharides, with a prolonged lag phase preceding the burst of thrombin generation. In thromboplastin-activated plasma, thrombin generation was inhibited by CMDBS and fucoidan, with a prolonged lag phase only in the presence of CMDBS. The data obtained with each polysaccharide, compared to those obtained with heparin (our study) and hirudin (published data), led to hypothesize that fucoidan could act, like heparin, by forming complexes with the inhibitor (although antithrombin (AT) in the case of heparin, and HCII for fucoidan), while CMDBS could act, like hirudin, by forming complexes with thrombin.

Anticoagulant activity of dextran derivatives.

Carboxymethyl dextran benzylamide sulfonate/sulfates (CMDBS) are synthetic polysaccharides with anticoagulant activity. We synthesized eight different highly substituted CMDBS and one CMDSu. We studied both their anticoagulant activity and the catalysis of thrombin (T) inhibition by heparin cofactor II (HCII) and antithrombin (AT) in the presence of these dextran derivatives relative to heparin and dextran sulfate (DXSu). The anticoagulant activity of CMDBS was due both to direct thrombin inhibition and to catalysis of thrombin inhibition by HCII. The anticoagulant and catalytic activities of CMDBS were related mainly to their molecular weight and sulfate content. The interaction of the dextran derivatives with thrombin does not involve the active site of the enzyme. A kinetic study showed that all the CMDBS exhibited higher affinity for thrombin than heparin did but lower affinity than DXSu did, suggesting that the benzylamide and sulfate groups potentiate the interaction between the dextran derivatives and thrombin. This study shows that the mechanism by which the dextran derivatives inhibit thrombin is original and is related to preferential interaction with thrombin; this both inhibits the clotting activity of the enzyme and increases the reaction rate of thrombin inhibition by HCII.

Epstein-Barr virus-dependent lymphoproliferative disease: critical role of IL-6.

Epstein-Barr virus (EBV)-induced lymphoproliferative disease (lpd) is a B cell neoplasm that affects patients who are immunosuppressed in the context of organ transplantation or HIV infection. A model for the aggressive form of this entity was generated by xenotransplantation of SCID mice with human peripheral blood leukocytes from individuals with prior contact with EBV. This model, where large B cell lymphoma occurs, was used to test the hypothesis that IL-6 has a major role in EBV-induced B cell tumorigenesis. IL-6 is known to differentiate B cells into immunoglobulin-secreting plasma cells and induce EBV replication, and xenochimeric animals have detectable serum levels of human IL-6. Human IL-6 inhibition with a neutralizing monoclonal antibody decreased tumor incidence from 62 % to 27 %. In addition, anti-IL-6 treatment significantly improved xenotransplanted animal survival, with median survival at > 245 days when compared to that of controls at 132 days. In conclusion, IL-6 plays a critical role in the pathogenesis of EBV-induced human lpd, and IL-6 inhibition may represent a new and promising preventive or therapeutic approach against this malignancy.