6)

6). not of N-degly-FX. Altogether our data lead to the conclusion that FX interaction with macrophages through its N-glycans protects it from a rapid clearance explaining its relatively long circulatory half-life. == Introduction == Human factor X (FX) is a vitamin K-dependent glycoprotein synthesized in the liver that circulates in plasma at a concentration LY315920 (Varespladib) of 10 g/mL as a two-chain zymogen protein. It is composed of the light chain containing a gamma-carboxyglutamic acid-rich domain or Gla domain followed by two epidermal growth factor (EGF)-like domains linked by a disulfide bond to the heavy chain. The heavy chain contains an activation peptide and a serine-protease domain. FX plays a central role in blood coagulation. During this process, FX is activated to FXa by proteolytic cleavage of the heavy chain and subsequent release of the 52 amino acid activation glycopeptide. This cleavage also leads to a rearrangement of the serine protease domain and the formation of the catalytic site of the enzyme. Subsequently, FXa forms a high affinity macromolecular complex with other components of the prothrombinase complex: Factor Va (FVa), negatively-charged phospholipid surfaces and calcium. This prothrombinase complex activates prothrombin to thrombin leading to the formation of a hemostatic plug and local hemostasis by conversion of fibrinogen into fibrin[1][5]. While the functions, structure, and structure-function relationships of FX and FXa have been widely studied, the clearance process of the circulating enzyme and particularly of the zymogen has remained largely unexplored. Strikingly, FX has a long survival (half-life of 40 hours) compared to closely related vitamin K-dependent coagulation factors like factors VII (5 hours), IX (1824 hours) and protein C (4 hours)[6][8]. In view of the promising therapeutic strategies based on modified FX molecules to treat hemophilia[9],[10], a better knowledge of FX clearance mechanisms appears necessary. Interestingly, FX clearance kinetics showed a slower clearance rate than that of FXa, which has a half-life reduced to only few minutes[11]. There are several structural differences between FXa and FX: among them the presence of an active catalytic site, and absence of the activation peptide in FXa compared to FX. The role of the catalytic site in FXa clearance has been investigated using inactivated FXa. Two groups showed that inactivation of the catalytic site did not prolong the half-life of FXa to the value observed of FX[11],[12]suggesting that the activation peptide domain of FX could play a role in FX clearance. This hypothesis has been investigated in two more recent studies. First, we have shown Rabbit Polyclonal to SLC9A9 that the carboxy-terminal LY315920 (Varespladib) end LY315920 (Varespladib) of the activation peptide plays a crucial role in FX catabolism, both in recovery and half-life of the protein[13]. Moreover, it was also shown that insertion of the FX activation peptide in FVII increased the terminal half-life of the latter 4-fold[14]. Interestingly, both studies demonstrated that the two sole N-linked glycans of FX at positions 181 and 191 of its activation peptide represent important structural determinants for the recovery and half-life of the injected protein. In view of these observations, the purpose of this study was to explore the mechanism that could explain the role of the Nglycosylation sites in FX clearance. To this end, we have compared removal pathways from the circulation of human plasma and recombinant FX (pd-FX and rFX) with N-deglycosylated plasma FX (N-degly-FX) and rFX mutated at both N-glycosylation sites (rFXN181AN191A), and attempted to determine organs and cell-types potentially involved in this process. == Materials and Methods == An extensive description of materials and methods can be found in theMaterials and Methods S1. == Mice == Wild-type mice C57Bl/6 were purchased from Janvier (Le Genest Saint Isle, France). Housing and experiments were done as recommended by French regulations and the experimental guidelines of the European Community. The Animal Care and Use Committee of INSERM approved animal experiments (licence #B-94-043-13). == Factor X Protein Preparation == Full-length Glycosidase digestion experiments under mild conditions on N-linked glycans ofplasma-derived factor X (pd-FX from Haematologic Technologies Inc, Vermont, USA)were carried out usingPNGase F (N-Glycosidase F from New England Biolabs Inc, Essex Junction, MA, USA)from Chryseobacterium as previously described[15]. Protein LY315920 (Varespladib) purity (>95%) and full removal of.