Anne-Marie Fischer

Evaluation of Prothrombin Complex Concentrate and Recombinant Activated Factor VII to Reverse Rivaroxaban in a Rabbit Model

Background: As a potent anticoagulant agent, rivaroxaban exposes a risk of bleeding. An effective way to reverse its effects is needed. Objectives were to study efficacy and safety of recombinant activated factor VII (rFVIIa) and prothrombin complex concentrate (PCC) to reverse the anticoagulant effect of an overdose of rivaroxaban in a rabbit model of bleeding and thrombosis. Methods: First, a dose-ranging study assessed the minimal rivaroxaban dose that increased bleeding. Then, 48 anesthetized and ventilated rabbits were randomized into four groups: control (saline), rivaroxaban (rivaroxaban and saline), rFVIIa (rivaroxaban and rFVIIa), and PCC (rivaroxaban and PCC). The Folts model was applied: a stenosis and an injury were carried out on the carotid artery, inducing thrombosis, detected as cyclic flow reductions, which were recorded over 20 min. Then the following were measured: ear immersion bleeding time, clotting times, anti-Xa activity, thrombelastometric parameters, and thrombin generation test. Ultimately, a hepatosplenic section was performed and the total amount of blood loss after 15 min was evaluated as primary endpoint. Results: Rivaroxaban increased blood loss (17 g [8–32] vs. 7 g [5–18] for control (median [range]), P = 0.0004), ear bleeding time, clotting times, thrombelastographic clotting time, and decreased thrombin generation. In contrast, rFVIIa decreased ear bleeding time (92 s [65–115] vs. 140 s [75–190], P < 0.02), but without efficacy on blood loss. PCC and rFVIIa decreased activated partial thromboplastin time as well as thrombelastographic clotting time. Regarding safety, neither rFVIIa nor PCC increased cyclic flow reductions. Conclusion: rFVIIa and PCC partially improved laboratory parameters, but did not reverse rivaroxaban induced-bleeding.

Anticoagulant properties of a fucoidan fraction

Fucoïdans are a family of high molecular weight sulphated polysaccharides in the Mr range 8 x 10(5) -10(6), widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata, was undertaken to obtain a low molecular weight polysaccharide (Mr 20,000 +/- 5,000) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor IIa and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoïdan revealed a potent antithrombin activity: studied in an antithrombin III depleted plasma or in the presence of purified heparin cofactor II, the fucoïdan was as efficient as heparin and dermatan sulphate on heparin cofactor II potentiation, at the same concentration by weight. In whole plasma or in the presence of the purified inhibitor, an anti-factor IIa activity mediated by antithrombin III was detected (30 times less potent than for heparin, on a weight to weight basis). In contrast, no anti-factor Xa activity was detected in the presence of the degraded fucoïdan, under the same experimental conditions. These fucoïdans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.

Marine Polysaccharides: A Source of Bioactive Molecules for Cell Therapy and Tissue Engineering

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

Evaluation of hemostasis and endothelial function in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab

Background Paroxysmal nocturnal hemoglobinuria (PNH) is associated with an increased risk of thrombosis through unknown mechanisms. Design and Methods We studied 23 patients with PNH, before and after five and 11 weeks of treatment with eculizumab. We examined markers of thrombin generation and reactional fibrinolysis (prothrombin fragment 1+2 (F1+2), D-dimers, and plasmin antiplasmin complexes (P-AP), and endothelial dysfunction tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), soluble thrombomodulin (sTM), intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule (sVCAM-1), endothelial microparticles (EMPs), and tissue factor pathway inhibitor (TFPI). Results At baseline, vWF, sVCAM-1, the EMP count, and F1+2 and D-dimer levels were significantly elevated in the patients, including those with no history of clinical thrombosis. Treatment with eculizumab was associated with significant decreases in plasma markers of coagulation activation (F1+2, P=0.012, and D-dimers, P=0.01), and reactional fibrinolysis (P-AP, P=0.0002). Eculizumab treatment also significantly reduced plasma markers of endothelial cell activation (t-PA, P=0.0005, sVCAM-1, P<0.0001, and vWF, P=0.0047) and total (P=0.0008) and free (P=0.0013) TFPI plasma levels. Conclusions Our results suggest a new understanding of the contribution of endothelial cell activation to the pathogenesis of thrombosis in PNH. The terminal complement inhibitor, eculizumab, induced a significant and sustained decrease in the activation of both the plasma hemostatic system and the vascular endothelium, likely contributing to the protective effect of eculizumab on thrombosis in this setting.

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.

Interactions of poly(lactic acid) and poly(lactic acid-co-ethylene oxide) nanoparticles with the plasma factors of the coagulation system

When surfactant-stabilized biodegradable poly(lactic acid) (PLA) particles are injected into rats, the rate of clearance from blood is fast. The rate can be strongly reduced by using particles made from diblock copolymers of PLA and poly(ethylene oxide) (PLA-PEO), resulting in an increased duration of contact with the components of the coagulation system. Thus, possible adverse effects such as activation of the coagulation cascade could occur. In this paper, the interactions of surfactant-stabilized PLA and PLA-PEO nanoparticle suspensions with the plasma factors of the coagulation system are presented. PLA suspensions stabilized by sodium cholate (PLA-Ch) interact with thrombin, factor V and calcium ions. Formation of complexes and aggregates is induced by addition of calcium ions to PLA-Ch suspensions in the presence or in the absence of plasma. On the contrary, PLA-PEO suspensions are remarkably inert towards the coagulation factors and calcium ions, even when cholate is present. Steric repulsion owing to the high surface density of PEO is sufficient to avoid strong interations with the proteins and formation of aggregates between particles.

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.

α6-Integrin Subunit Plays a Major Role in the Proangiogenic Properties of Endothelial Progenitor Cells

Objective—Alpha6 integrin subunit (&agr;6) expression is increased by proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor. This increase correlates with enhanced in vitro tube formation by endothelial cells and their progenitors called Endothelial Colony-Forming Cells (ECFCs). We thus studied the role of &agr;6 in vasculogenesis induced by human ECFCs, in a mouse model of hindlimb ischemia. Methods and Results—We used small interfering RNA (siRNA) to inhibit &agr;6 expression on the surface of ECFCs. For in vivo studies, human ECFCs were injected intravenously into a nude mouse model of unilateral hind limb ischemia. Transfection with siRNA &agr;6 abrogated neovessel formation and reperfusion of the ischemic hind limb induced by ECFCs (P<0.01 and P<0.001, respectively). It also inhibited ECFC incorporation into the vasculature of the ischemic muscle (P<0.001). In vitro, siRNA &agr;6 inhibited ECFC adhesion (P<0.01), pseudotube formation on Matrigel, migration, and AKT phosphorylation (P<0.0001), with no effect on cell proliferation or apoptosis. Conclusion—&agr;6 Expression is required for ECFC migration, adhesion, recruitment at the site of ischemia, and the promotion of the postischemic vascular repair. Thus, we have demonstrated a major role of &agr;6 in the proangiogenic properties of ECFCs.

Severe hypofibrinogenemia associated with bilateral ischemic necrosis of toes and fingers.

Severe hypofibrinogenemia was found in an Algerian woman who, since the age of 37 years, suffered three different episodes of ischemic necrosis of the toes and fingers leading to amputation of the toes and surgical removal of necrotic tissue (necretomy). No anti-fibrinogen antibody was present. The deficiency appeared to be due to severe congenital hypofibrinogenemia since the fibrinogen level remained at the same low level over a long period, without any abnormality of other coagulation proteins. The thrombotic events may be explained by the increased thrombin generation observed in the patients plasma, due to the lack of thrombin adsorption onto a fibrin net.

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.