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Activation of extracellular-signal-regulated protein kinase (Erk) is central to growth-factor-receptor-mediated signaling including that originating from the T cell antigen receptor. It integrates cytoplasmic signals to effect changes in transcription associated with differentiation, proliferation, and survival. In this report, we present an analysis of mice with targeted deletions in Erk1 and Erk2 to assess the relationship between Erk activity and cell-cycle progression, thymocyte development, and lineage commitment. These studies show that Erk is selectively retained during beta selection-driven proliferation, and yet Erk1/2 are not required to complete differentiation to CD4+CD8+ preselection stage of development. Erk activity is essential for the process of positive selection, and it differentially affects CD4 and CD8 T cell maturation; yet, diminished expression itself is not sufficient to alter lineage commitment.

Antibodies raised against tonoplast intrinsic proteins (TIPs) were used to probe the functional status of the soybean [Glycine max (L.) Merr.] paraveinal mesophyll (PVM) vacuole during changes in nitrogen metabolism within the leaf. Young plants grown under standard conditions had PVM vacuoles characterised by the presence of γ-TIP, which is indicative of a lytic function. When plants were then subjected to shoot tip removal for a period of 15 d, forcing a sink-limited physiological condition, the γ-TIP marker diminished while the δ-TIP marker became present in the PVM vacuole, indicating the conversion of the PVM vacuole to a storage function. When the shoot tips were allowed to regrow, the γ-TIP marker again became dominant demonstrating the reversion of these PVM vacuoles back to a lytic compartment. The changes in TIP markers correlated with the accumulation of vegetative storage proteins and vegetative lipoxygenases, proteins implicated in nitrogen storage and assimilate partitioning. This research suggests that the PVM vacuole is able to undergo dynamic conversion between lytic and storage functions and further implicates this cell layer in assimilate storage and mobilisation in soybeans.

The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to alpha(6) integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-alpha(6) blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2 and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.

We describe here five F7 mutations found in four patients without bleeding history, despite constitutional coagulation Factor VII (FVII) deficiency. All five mutations are missense and affect the catalytic domain of FVII (A191T, A191V, T239P, R224Q and M298I). The A191V and T239P mutations are novel and were found in homozygous patients with no clinical bleeding tendency. The patient diagnosed with the A191V mutation had a phenotype corresponding to a moderate type 1 FVII deficiency (FVII:C 4%, FVII:Ag 5%). The T239P mutation was found in a patient with mild type 2 FVII deficiency (FVII:C 25%, FVII:Ag 95%). Novel mutations are both in close vicinity to the charge-stabilizing system of FVII. Modeling studies allow understanding in part the molecular basis for the loss of function.

Most of the nitrogen harvested with the seeds of annual crops is remobilized and retranslocated within the plant between anthesis and plant death. While chloroplasts contain most of the reduced nitrogen present in photosynthetically active leaf cells, the (major) pathway(s) involved in the degradation of their proteins prior to the retranslocation of the resulting amino acids are unknown. In this study, a population of 146 recombinant inbred barley lines (RIL), derived from the cross between two varieties with a highly inheritable difference in grain protein concentration, was used to map quantitative trait loci (QTL) for leaf amino-, carboxy- and endopeptidase activities relative to previously determined QTL for grain protein, leaf N storage, and remobilization. The results strongly suggested that major endopeptidases, assayed at both acidic and slightly alkaline pH values (favouring vacuolar and extravacuolar enzymes, respectively) are not instrumental in leaf N remobilization or the control of grain protein accumulation. Similarly, QTL determined for aminopeptidases (relative to QTL for N remobilization) indicated no functional role for the enzyme(s) assayed in plant N recycling. By contrast, careful evaluation of QTL data suggested that one or several carboxypeptidase isoenzymes may be involved in this physiologically and economically important process. As these proteases (in contrast to aminopeptidases) have previously been localized in vacuoles, this result appears intriguing. These data, while shedding new light on an old problem, also indicate that the described approach may prove useful in evaluating the functional roles of additional (not assayed in this study) proteolytic systems in whole-plant nitrogen recycling.

Chemokines are critical in controlling lymphocyte traffic and migration. The CXC chemokine CXCL12/SDF-1alpha interacts with its receptor CXCR4 to induce the migration of a number of different cell types. Although an understanding of the physiological functions of this chemokine is emerging, the mechanism by which it regulates T cell migration is still unclear. We show here that the Tec family kinase ITK is activated rapidly following CXCL12/SDF-1alpha stimulation, and this requires Src and phosphatidylinositol 3-kinase activities. ITK regulates the ability of CXCL12/SDF-1alpha to induce T cell migration as overexpression of wild-type ITK-enhanced migration, and T cells lacking ITK exhibit reduced migration as well as adhesion in response to CXCL12/SDF-1alpha. Further analysis suggests that ITK may regulate CXCR4-mediated migration and adhesion by altering the actin cytoskeleton, as ITK null T cells were significantly defective in CXCL12/SDF-1a-mediated actin polymerization. Our data suggest that ITK may regulate the ability of CXCR4 to induce T cell migration.

Basic fibroblast growth factor (FGF-2) activates its high-affinity receptors (FGFRs) but also acts through interaction with heparan sulfate proteoglycans (HSPG). Exogenous polysaccharides also modulate the angiogenic activity of FGF-2. We investigated the effect and mechanism of action of a low molecular weight fucoidan derivative (LMWF) on tube formation by human endothelial cells. LMWF has a better arterial antithrombotic potential in animals than low molecular weight heparin (LMWH). After stimulation of human umbilical vein endothelial cells (HUVEC) by FGF-2 and LMWF (or LMWH), we observed 1) using flow cytometry, an increase in the amount of the alpha6 integrin subunit; 2) using quantitative reverse transcription-polymerase chain reaction, an increase in alpha6 mRNA (higher with LMWF than with LMWH); and 3) using a Matrigel model, an increase in vascular tube formation (also higher with LMWF than with LMWH). A direct link between alpha6 overexpression and vascular tube formation was confirmed by use of an anti-alpha6 antibody: in its presence, there was no capillary network formation on Matrigel. Unexpectedly, an anti-FGFR blocking antibody had no effect on alpha6 over-expression, whereas stripping off the heparan sulfate with heparitinases abolished overexpression. Overall, our data suggest that FGF-2 stimulates alpha6 over-expression in HUVEC, through HSPG but independently from FGFR, and that LMWF (or LMWH) modulates this interaction. Expression of heparan sulfate proteoglycan increases after ischemic injury. Given its antithrombotic properties and its ability to potentiate tube formation of endothelial cells, LMWF may have to be considered for revascularization of ischemic areas.

In a patient with lethal factor VII (FVII) deficiency, 2 homozygous nucleotide substitutions were identified in the F7 gene: a IVS7+2T>G transversion involving the IVS7 donor splice site, followed by a mutation at nucleotide 10588 that would result in a missense variation (Arg224Gln). The mutated splice site, located within the first repeat of a minisatellite, is followed by a variable number of pseudo-sites, normally silent. To investigate the consequences of this mutation on F7 splicing, we designed normal and mutant minigenes, spanning exons 5 to 8. In cells transfected with the mutant construct, no normal splicing occurred. Only spliced transcripts including the first minisatellite repeat were observed, resulting from the activation of the most proximal wild-type pseudo-site, which would generate a truncated protein (stop codon upstream of nucleotide 10588). These findings, which suggest the existence of a mechanism selecting one single splice site among multiple cryptic sites, explain the patient's phenotype.

In this work we test the hypothesis that yield of rice ( Oryza sativa L.) can be enhanced by increasing endosperm activity of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch biosynthesis. The potential for increases in yield exist because rice initiates more seeds than are taken to maturity and possesses excess photosynthetic capacity that could be utilized if there were more demand for assimilate. Following an approach already shown to be successful in wheat, experiments were designed to increase demand for assimilate by increasing the capacity for starch synthesis in endosperm. This was accomplished by transforming rice with a modified maize AGP large subunit sequence ( Sh2r6hs) under control of an endosperm-specific promoter. This altered subunit confers upon AGP decreased sensitivity to allosteric inhibition by inorganic phosphate (Pi) and enhanced heat stability, potentially leading to higher AGP activity in vivo. The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. Increases in AGP activity in transgenic seeds compared with controls were maximal between 10-15 days after anthesis. Starch content of individual seeds at harvest was not increased, but seed weight per plant and total plant biomass were each increased by more than 20%. Increased endosperm AGP activity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set. Results demonstrate that deregulation of endosperm AGP increases overall plant sink strength, leading to larger, more productive plants in a manner similar to that in wheat having similar genetic modification.

Nitrogen uptake and metabolism are central for vegetative and reproductive plant growth. This is reflected by the fact that nitrogen can be remobilized and reused within a plant, and this process is crucial for yield in most annual crops. A population of 146 recombinant inbred barley lines (F(8) and F(9) plants, grown in 2000 and 2001), derived from a cross between two varieties differing markedly in grain protein concentration, was used to compare the location of QTL associated with nitrogen uptake, storage and remobilization in flag leaves relative to QTL controlling developmental parameters and grain protein accumulation. Overlaps of support intervals for such QTL were found on several chromosomes, with chromosomes 3 and 6 being especially important. For QTL on these chromosomes, alleles associated with inefficient N remobilization were associated with depressed yield and higher levels of total or soluble organic nitrogen during grain filling and vice versa; therefore, genes directly involved in N recycling or genes regulating N recycling may be located on these chromosomes. Interestingly, the most prominent QTL for grain protein concentration (on chromosome 6) did not co-localize with QTL for nitrogen remobilization. However, QTL peaks for nitrate and soluble organic nitrogen were detected at this locus for plants grown in 2001 (but not in 2000). For these, alleles associated with low grain protein concentration were associated with higher soluble nitrogen levels in leaves during grain filling; therefore, gene(s) found at this locus might influence the nitrogen sink strength of developing barley grains.

Fucosylated chondroitin sulfate (FucCS), a glycosaminoglycan obtained from sea cucumber, has the same structure as mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. This new polysaccharide has a more favorable effect than heparin on vascular cell growth. It inhibits smooth muscle cell proliferation as heparin, and it has a potent enhancing effect on endothelial cell proliferation and migration in the presence of heparin-binding growth factors. We now extend our studies to the effect of this glycosaminoglycan on endothelial cells to an in vitro angiogenesis model on Matrigel. FucCS, in the presence of fibroblast growth factor-2 (FGF-2), strongly increases the capacity of endothelial cells to form vascular tubes on Matrigel with a well-organized capillary-like network and typical closed structures. Comparison between the activity of native and chemically modified chondroitin sulfate from sea cucumber reveals that the sulfated fucose branches are the structural motif for the proangiogenic activity. Heparin does not induce angiogenesis in this experimental model. We also have evidence for the proposition that endothelial cell proliferation is not the sole event involved in the in vitro FGF-2-induced angiogenesis. It implies a variety of other modifications of the endothelial cells and of their interaction with the extracellular matrix, such as integrin expression and actin cytoskeleton reorganization. Finally, the proangiogenic effect of FucCS, concomitant with its capacity to prevent venous and arterial thrombosis, in animal models makes this new glycosaminoglycan a promising molecule with possible beneficial effects in pathological conditions affecting blood vessels such as the neovascularization of ischemic areas.

Fucoidans are sulfated polysaccharides extracted from brown marine algae. A purified fucoidan fraction exhibits the same venous antithrombotic activity as heparin in rabbits, but with a lower anticoagulant effect. Because of its heparin-like structure, we postulated that fucoidan might modulate heparin-binding angiogenic growth factor activity. We thus studied its effect, at antithrombotic concentrations, on fibroblast growth factor (FGF)-2-induced proliferation and differentiation of human umbilical vein endothelial cells. The fucoidan effect on endothelial cell differentiation was evaluated by studying the expression of surface proteins (i.e. integrin, adhesion molecule) known to be modulated by FGF-2 and involved in angiogenesis, and by quantifying closed areas delimited by vascular tubes formed on reconstituted basement membrane. Fucoidan had no modulatory effect on the mitogenic activity of FGF-2, but significantly increased tubular structure density induced by FGF-2. Fucoidan alone increased alpha(6) integrin subunit expression with only partially organized tubular structure. In the presence of FGF-2, fucoidan enhanced alpha(6), beta(1) and PECAM-1 and inhibited alpha(v)beta(3) integrin expression. Heparin had no effect in these systems. The most striking effect of fucoidan was observed on alpha(6) expression and tube formation was abolished by monoclonal anti-alpha(6) antibodies. Fucoidan plus FGF-2 effect on alpha(6) expression was markedly decreased by monoclonal anti-FGF-2 antibodies, indicating that fucoidan acts mainly via FGF-2. These results show that, at antithrombotic concentrations, contrary to heparin, fucoidan can enhance vascular tube formation induced by FGF-2 with a modulation of the expression of surface proteins (mainly alpha(6)) involved in angiogenesis.

The molecular basis of severe type I factor (F)VII deficiency was investigated in two Algerian patients. One patient, a 13-year-old-girl who has suffered from severe bleeding since birth, was homozygous for a 7-bp deletion (nt 7774-7780) and a 251-bp insertion (nt 7773-7781) of mitochondrial origin, in IVS 4 acceptor splice site. The other patient, an infant who died from massive intracranial haemorrhage, was homozygous for a transversion in the IVS 7 donor splice site (T9726+2-->G) and a missense mutation in exon 8 (G10588-->A; Arg224-->Gln). In both cases, the deleterious mutations are probably the splice site junction abnormalities impairing mRNA processing. These three lesions have not yet been reported.

A new low-molecular-weight 'heparin-like' component was obtained from an exopolysaccharide produced by a mesophilic strain found in deep-sea hydrothermal vents. Data concerning the structure of the native high-molecular-weight exopolysaccharide (10(6) g/mol, 10% sulfate content) are reported for the first time. Two depolymerization processes were used to obtain low-molecular-weight (24-35x10(3) g/mol) oversulfated fractions (sulfate content 20 or 40%). Nuclear magnetic resonance studies indicated that after sulfation (40%), the low-molecular-weight fraction obtained by free radical depolymerization was less sulfated in the 6-O-position than the fraction depolymerized by acid hydrolysis. The free radical depolymerized product also had sulfated residues in the 4-O-position and disulfated ones in the 2,3-O-positions. Moreover, the compounds generated by the free radical process were more homogeneous with respect to molecular mass. Also for the first time, the anticoagulant activity of the low-molecular-weight exopolysaccharide fractions is reported. When the fractions obtained after sulfation and depolymerization were compared with heparins, anticoagulant activity was detected in oversulfated fractions, but not in native exopolysaccharide. The free radical depolymerized fraction inhibited thrombin generation in both contact-activated and thromboplastin-activated plasma, showing a prolonged lag phase only in the contact-activated assay. Affinity co-electrophoresis studies suggested that a single population of polysaccharide chains binds to antithrombin and that only a subpopulation strongly interacts with heparin cofactor II.

In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present. Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning. In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coil. The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and alpha-linolenic acid, and the reaction products with the substrates linoleic acid, alpha-linolenic acid, arachidonic acid, gamma-linolenic acid, and the triacylglycerol trilinolein. All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid. The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols. Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf. Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed.

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 +/- 5 microg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 microg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 +/- 7.2%) and 27% (126.9 +/- 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 microg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 +/- 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 +/- 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form. +98% (198.2 +/- 25%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

Plasma levels of prothrombin fragment 1+2 (Fl+2), thrombin-antithrombin complexes (TAT) and D-dimers were measured in 15 patients with pulmonary embolism during heparin therapy, oral anticoagulation, and after cessation of warfarin therapy. Each patient had a favorable outcome during anticoagulant therapy (3 months), but late venous thromboembolism occurred in six cases. The mean levels of the three markers were significantly increased on day 4 after the thrombotic event, and normalized during warfarin therapy. Nine months after the initial pulmonary embolism, mean levels of the three markers, as compared with a control population, were significantly higher in the patients with late recurrences, whereas only TAT were slightly higher in patients without recurrences as compared with controls. Only TAT levels were significantly higher in the patients with late recurrences than in those without late recurrences. Thus, the levels of the three markers 9 months after pulmonary embolism seem to be interesting to identify patients with high risk of recurrence and who might require longer anticoagulant treatment.

Vegetative lipoxygenases (VLXs) in soybean are hypothesized to function in nitrogen storage and partitioning. Isoform-specific antibodies for four of the five known VLX isoenzymes were used to investigate the influence of source-sink status on protein levels, as well as to analyze the tissue and subcellular localization of the different isoforms. VLXD responded most strongly to sink limitation, although the levels of VLXA, B and C increased as well. After sink limitation, VLXD and the vegetative storage protein, VSPalpha, accumulated in the vacuoles of bundle sheath and paraveinal mesophyll cells, while VLXA, B and C localized to the cytosol of these cells. All five known VLX isoenzymes were active with both linoleic and linolenic acid substrates after expression in Escherichia coli. The strong upregulation of VLXD levels after sink limitation as well as the localization of this isoform to the vacuoles of paraveinal mesophyll and bundle sheath cells (where VSPs are found) strongly suggest that VLXD should be considered as a major storage protein in soybean leaves. Furthermore, since VLXA, B and C also accumulate in sink-limited soybean leaves, are located in the cytosol of paraveinal mesophyll cells and are active at pH values typically found in this compartment, their activities may well contribute to lipid metabolism in this tissue. This multi-gene family is thus ideally poised to play a pivotal role in the balance of N deposition relative to lipid-based storage, defense or signaling, by modulating contributions to these processes in the transient storage cells of the paraveinal mesophyll.

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.

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.

Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), delta-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of delta-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity.

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has antithrombotic properties, the mechanism of which is not yet completely understood. Tissue factor pathway inhibitor (TFPI), which regulates the tissue factor-dependent pathway of blood coagulation, is released from the endothelium by heparin, a mechanism contributing to its antithrombotic activity. In this study, we demonstrated that fucoidan, as heparin, induces TFPI release from cultured human umbilical vein endothelial cells (HUVEC). The TFPI accumulation in the HUVEC supernatants depends on the incubation time and polysaccharide concentration. After 30 to 60 minutes of incubation, TFPI concentration (total antigen level) was twice higher in the presence of both polysaccharides than in their absence. After one hour of incubation, in the presence of increasing concentrations of each polysaccharide, an optimal stimulation was observed for 0.5 microg/ml of fucoidan and 5 microg/ml of heparin, as evidenced by a raise of the basal TFPI level: a 2-fold increase for the total antigen and a 3-fold increase for the free antigen. These data suggest that TFPI released from vascular endothelial cells may contribute to the antithrombotic effect of fucoidan.

A semiquantitative enzyme-linked immunosorbent assay (ELISA) test (Instant IA D-dimer) and a new quantitative rapid and individual test (STA-Liatest DDi) were compared with the reference microplate ELISA (Asserachrom D-Di) for D-dimer testing in 142 patients clinically suspected of pulmonary embolism, on the basis of clinical symptoms and signs, electrocardiogram, blood gases and chest X-Ray abnormalities. The cut-off value for the quantitative tests was 500 ng/ml and Instant IA was interpreted by three readers. Pulmonary embolism was confirmed by lung scan or angiography in 60 patients (42%). The sensitivities of ELISA and STA-Liatest DDi were 92% [95% confidence interval (CI) 82-97%] and 93% (95% CI 84-98%), respectively. The three readings of Instant IA D-dimer disagreed in 27 (19%) of the patients and sensitivity varied from 83 to 93% according to the readers. In the 115 patients with concordant readings, sensitivity was 92% (95% CI 82-98%). These results suggest that STA Liatest DDi may be used instead of microplate ELISA for the exclusion of pulmonary embolism, whereas the use of Instant IA D-dimer for this purpose is limited by the number of discordant results.

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.

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.

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.

The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from 10(-9) to 10(-4) M. The initial concentrations of inhibitor (l) and thrombin (E) were set at equimolar levels (CI = CE = 10(-8) M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from 10(-9) to 10(-6) M and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902-2913] for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. KH,I, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 10(-7) M and 1.3 x 10(9) M-1 min-1, respectively, for AT, compared to a KH,I of 2.0 x 10(-6) M and k of 6.4 x 10(9) M-1 min-1 for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10(-6) M, the decrease in the reaction rate was in keeping with the formation of a heparin-thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of the free protease.

The kinetics of thrombin inhibition by heparin cofactor II (HC II) in the presence of dermatan sulphates, native (DS), or oversulphated (DSS 1 and DSS 2) and a biospecific dextran derivative substituted with carboxymethyl, carboxymethyl-benzylamide and carboxymethyl benzylamide-sulphonate functional groups (CMDBS), has been studied as a function of the sulphated polysaccharide concentration. The initial HC II and thrombin concentrations were set at equimolar levels. Analysis of the experimental data obtained for DS, DSS1 and DSS2 was performed using a previously described model which allows computation of the dissociation constant (KPS,HC) of the polysaccharide-HC II complex and the rate constant of thrombin inhibition by the polysaccharide-HC II complex (k). A KPS.HC of 9.6 x'10(-7) M and a k of 4.5 x 10(9) M-1 min-1 were found for DS, whereas KPS,HC 2.1 x 10(-6) M, k 1.1 x 10(10) M-1 min-1 and KPS,HC 4.3 x 10(-7) M, k 1.4 x 10(10) M-1 min-1 were found for DSS1 and DSS2, respectively. Knowing that DSS1 has a sulphur content per disaccharide of 7.8%, compared with 11.5% for DSS2, these results indicate that the polysaccharide affinity for HC II is increased only in the case of DSS 2, whereas the oversulphation increases the reactivities towards thrombin of both complexes DSS1-HC II and DSS2-HC II. A better conformation of these complexes may favour a faster interaction with the protease. Unlike heparin, DS at concentrations higher than 10(-5) M does not modify the reaction rate of thrombin inhibition, a fact which can be explained by the absence of complex formation between DS and thrombin. The experimental data obtained for CMDBS fit a kinetic model in which the biospecific dextran derivative rapidly forms a complex with thrombin which is more reactive towards HC II than the free protease. The reaction rate remained unchanged for CMDBS concentrations equal to or higher than 10(-5) M, whereas CMDBS was found to interfere strongly with the fibrinogen-thrombin interaction. These data suggest that CMDBS has a strong affinity for the protease and no affinity for HC II. The computed dissociation constant of the CMDBS-thrombin complex (KPS,E) was 2.4 x 10(-7) M and the rate constant of the reaction of this complex with HC II (k) was 1.7 x 10(8) M-1 min-1. These findings indicate that CMDBS exerts its catalytic effect through a unique mechanism of action and may constitute a new class of anticoagulant drugs.

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.

Heparin-induced thrombocytopenia is very uncommon with low-molecular-weight heparin, especially when given for prophylaxis of venous thromboembolism. In two of the four published cases, with thrombotic complications, thrombocytopenia may have been related to cross-reactivity between unfractionated heparin and low-molecular-weight heparin. We report one patient who received low-molecular-weight heparin for prophylaxis against venous thromboembolism without any previous injection of unfractionated heparin, and experienced thrombocytopenia with thrombotic complications. Heparin-induced thrombocytopenia was confirmed by several laboratory assays. This observation emphasizes the need for platelet count monitoring during low-molecular-weight heparin therapy.

Thrombus formation on blood-contacting artificial surfaces is a major problem. Antithrombogenic polymer surfaces have been obtained either by heparin binding, or by grafting sulphonate and/or amino acid sulphonamide groups on insoluble polystyrene. In addition to their capacity to adsorb thrombin, such surfaces were shown to be able to catalyse its inhibition by antithrombin III (AT), i.e. they are endowed with heparin-like activity. The results were mainly obtained by using clotting assays. In many cases, delineating adsorption and catalytic processes by such assays is not possible when evaluating anticoagulant polymer surfaces. To overcome this problem, the kinetics of thrombin adsorption and inhibitions by AT and heparin cofactor II (HC) in the presence of such surfaces have been measured by using an assay performed with a thrombin-specific chromogenic substrate. A simple kinetic model of thrombin consumption is proposed. The relevant calculations, carried out with the help of a computer program, lead to determination of relative second order rate constants of thrombin adsorption and inhibitions by AT and HC in the presence of the polymers. In addition to thrombin adsorption, polystyrene surfaces bearing only sulphonate groups catalyse inhibition by AT, whereas polystyrene surfaces bearing either aspartate, glycinate or isophthalate sulphonamide groups catalyse both inhibitions by AT and HC.

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.

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 patient's plasma, due to the lack of thrombin adsorption onto a fibrin net.

This study reports on the biological data of ten patients with acute venous thrombo-embolism. They were treated for 5 days with continuous intravenous infusion of a fixed dose (0.05 mg/kg/hr) of a recombinant hirudin (r-H HBW 023 Behringwerke, Germany). The plasma level of r-H (HBW 023), assessed by an anti-factor IIa amidolytic activity, was stable after Day 2 and showed considerable individual variations. It correlated with APTT ratio, suggesting that this test is a reliable tool to monitor therapy. In contrast, thrombin time was constantly over 120 sec (control 15 sec) and consequently was not a useful parameter. Prothrombin time showed a slight, but significant, prolongation, which was correlated with the increase of APTT ratio. There was no bleeding time prolongation, platelet count, or ATIII level decrease. Levels of thrombin-antithrombin III complexes, and D-dimers, which were high in all patients on admission, decreased during the course of the treatment but remained abnormal on Day 5, showing an ongoing hemostasis and fibrinolysis activation: this is consistent with the delayed, but only slightly decreased thrombin generation evidenced by thrombin generation test performed on Day 3. These results suggest that thrombin inhibition by rH-hirudin at this dosage is only partial, which allows the generation of traces of thrombin needed for the feed-back thrombin production generated by factor V and VIII activation.

Isolated acquired factor VII (FVII) deficiency (0.15 U/ml) was identified in a 30-year-old man with pleural liposarcoma. The patient underwent surgery with continuous FVII concentrate infusion. No anti-FVII antibody or FVII/anti-FVII complex was detected. However, the short half-life and low recovery of FVII after concentrate infusion suggested the presence of an antibody. Whatever the mechanism, this FVII deficiency was related to the presence of the liposarcoma. FVII level normalized during tumour regression and fell again when the liposarcoma relapsed.

Three sulphated polysaccharides, dermatan sulphate, fucan and heparin, were fractionated according to their affinity towards antithrombin III (ATIII) and heparin cofactor II (HCII), the two main physiological thrombin (IIa) inhibitors. Both inhibitors were immobilized on concanavalin A-Sepharose, which binds to the glycosylated chains of the proteins while the protein-binding site for the polysaccharide remains free. Each polysaccharide was fractionated into bound and unbound fractions either for ATIII or HCII. The eluted fractions were tested for their ability to catalyse ATIII/IIa and HCII/IIa interactions. The possible presence of a unique binding site for ATIII and HCII, on each sulphated polysaccharide, was also studied.

Biospecific functional polymers, i.e., polymers randomly substituted with specific chemical functional groups, were designed to interact with living systems. Interactions between polystyrene sodium sulfonate (PSSO3Na) and insulin secreting RINm5F cells have been previously described. For the sake of comparison, interactions of PSSO3Na with human umbilical vein endothelial cells (HUVEC) were studied. In this case, the interaction is indirect, i.e., mediated by a binding protein, fibronectin (Fn). This was evidenced by HUVEC culture on Fn precoated PSSO3Na microcarriers. The interactions between PSSO3Na and HUVEC result in a biologically normal proliferation of cells and synthesis and secretion of Von Willebrand Factor (VWF). These results show that different biospecific interactions may occur between cells in culture, binding proteins and polymers randomly substituted with suitable functional groups. HUVEC, when cultured on heparin-like microcarriers, behave differently from other cells like RINm5F, whose interaction with the same polymers is not mediated by binding proteins.

Severe combined immunodeficiency (scid) mice develop EBV (+)B cell tumors after infusion of EBV(+)B cells or of B cells and EBV. In this study, scid mice were infused with B cell lines derived from three patients who developed a B lymphocyte proliferative disorder after bone marrow or organ transplantation. Intraperitoneal injection of 5 x 10(6) B cells induced tumor growth in all mice, leading to death within 60 d. Human B cells were identified in spleen and bone marrow by means of immunofluorescence or EBV genome amplification, and human IgM was detected in serum. Infusion of murine monoclonal antibodies specific for human B cell membrane antigens CD21, CD24, and CD23 was effective in 80% of animals, against two of the three cell lines preventing tumor development or inducing remission according to the time of treatment. The effect was antibody dose dependent and was optimal with four intravenous infusions of at least 0.1 mg 4 d apart. Human IgM in serum and human B cells in spleen and bone marrow became undetectable when peritoneal tumors regressed completely. Infusions of IgG1 isotype-matched anti-CD4 antibody or anti-CD3 antibody had no effect. Tumors developed or recurred in 50% of these animals injected with one of the B cell line 3 mo after treatment was stopped. The same anti-CD21 and anti-CD24 antibodies had been used to treat the three patients, and shown similar degrees of effectiveness as in the scid mouse model. These results indicate that scid mice may be suitable for assessing therapeutic approaches to human B cell proliferation.