Endothelial cell surface limits coagulation without modulating the antithrombin potency.

Antithrombin (AT) binds in vitro and in vivo to endothelial cells through various receptors, including heparan sulphate glycosaminoglycan (HSPG) that could modulate the AT activity. A thrombin generation assay (TGA) was set up at the surface of HUVEC and HMVEC evaluating their participation in the coagulation-anticoagulation processes. TGA induced by 0.5 pM Tissue Factor was performed in normal or AT-deficient plasma spiked with various amounts of recombinant or plasma-derived AT (0, 0.1, 0.5 and 1.0 U/ml). To evaluate the role of HSPG or cellular anticoagulant receptors, cells were treated or not with heparin, a mix of heparanase I, II and III, a neutralizing anti-Endothelial Protein C Receptor (EPCR) or with an anti-Tissue Factor Pathway Inhibitor (TFPI) antibody. The presence of the cells diminished the TG in normal plasma and maintained anticoagulation in AT-deficient plasma. Spiking the AT-deficient plasma with different doses of AT demonstrated that the cells did not amplify the anticoagulant activity of AT. The recombinant AT binds the cells with a higher avidity than the plasma-derived one but this did not affect its anticoagulant potency. Moreover both bindings are independent of the HSPG. The antithrombotic activity kept in absence of AT was not inhibited by blocking antibodies directed against EPCR or TFPI. Our data did not reveal a major co-factor activity for AT from endothelial cells that could have been mediated by HSPG. In contrast, it reveals the presence of alternative anti-coagulant system(s) in two venous cell types that maintain an antithrombotic activity.

The α1,6-fucosyltransferase gene (fut8) from the Sf9 lepidopteran insect cell line: insights into fut8 evolution.

The core alpha1,6-fucosyltransferase (FUT8) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the innermost asparagine-linked N-acetylglucosamine residue of glycoproteins. In mammals, this glycosylation has an important function in many fundamental biological processes and although no essential role has been demonstrated yet in all animals, FUT8 amino acid (aa) sequence and FUT8 activity are very well conserved throughout the animal kingdom. We have cloned the cDNA and the complete gene encoding the FUT8 in the Sf9 (Spodoptera frugiperda) lepidopteran cell line. As in most animal genomes, fut8 is a single-copy gene organized in different exons. The open reading frame contains 12 exons, a characteristic that seems to be shared by all lepidopteran fut8 genes. We chose to study the gene structure as a way to characterize the evolutionary relationships of the fut8 genes in metazoans. Analysis of the intron-exon organization in 56 fut8 orthologs allowed us to propose a model for fut8 evolution in metazoans. The presence of a highly variable number of exons in metazoan fut8 genes suggests a complex evolutionary history with many intron gain and loss events, particularly in arthropods, but not in chordata. Moreover, despite the high conservation of lepidoptera FUT8 sequences also in vertebrates and hymenoptera, the exon-intron organization of hymenoptera fut8 genes is order-specific with no shared exons. This feature suggests that the observed intron losses and gains may be linked to evolutionary innovations, such as the appearance of new orders.