title: Factor XIa dimer in the activation of factor IX.
abstract: Factor XI, unlike other coagulation proteins, is a homodimer of two identical subunits linked by a single disulfide bond formed by Cys321. The present study was undertaken to understand the physiological significance of the dimeric nature of factor XI. We have expressed a mutant FXI/G326C in which the Gly326 residue of factor XI has been mutated to Cys326, reasoning that Cys321 would form an intrachain disulfide bond with Cys326 as in prekallikrein, a plasma protein that exists as a monomer even with 58% amino acid sequence identity and a domain structure very similar to factor XI. No free thiol could be detected in the expressed protein, and it migrated as a monomer on nonreduced SDS-PAGE. In physiological buffer, however, the protein was found to exist in a state of monomer-dimer equilibrium as assessed by gel-filtration chromatography and ultracentrifugation studies (K(d) approximately 36 nM). Functional studies revealed that FXI/G326C was indistinguishable from plasma factor XI in a plasma-clotting assay and in a factor IX activation assay both in the presence and absence of activated platelets even at concentrations at which less than 5% of the mutant exists as dimers. We conclude that, for optimal function in the presence of activated platelets, a preformed dimer of factor XI is not required.
authors.affiliation: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. dipali@temple.edu
authors.forename: Dipali
authors.initials: D
authors.lastname: Sinha
authors.forename: Mariola
authors.initials: M
authors.lastname: Marcinkiewicz
authors.forename: James D
authors.initials: JD
authors.lastname: Lear
authors.forename: Peter N
authors.initials: PN
authors.lastname: Walsh
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D003545:Cysteine / Q000235:genetics; D016264:Dextran Sulfate / Q000494:pharmacology; D019281:Dimerization; D005164:Factor IX / Q000737:chemistry / Q000378:metabolism*; D015956:Factor XIIa / Q000502:physiology; D015945:Factor XIa / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005998:Glycine / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D009842:Oligopeptides / Q000378:metabolism; D010314:Partial Thromboplastin Time / Q000379:methods; D015539:Platelet Activation / Q000235:genetics; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013917:Thrombin / Q000502:physiology
chemical_list: D009842:Oligopeptides; C040190:S 2366; D005164:Factor IX; D016264:Dextran Sulfate; D015945:Factor XIa; D015956:Factor XIIa; D013917:Thrombin; D003545:Cysteine; D011761:Pyrrolidonecarboxylic Acid; D005998:Glycine