title: Blood coagulation factor IX-binding protein from the venom of Trimeresurus flavoviridis: purification and characterization.
abstract: The coagulation factor IX/factor X-binding protein (IX/X-bp) from the venom of Trimeresurus flavoviridis is a heterogeneous two-chain protein, and the structure of each chain is similar to that of the carbohydrate-recognition domain of C-type lectins, such as asialoglycoprotein receptors, pancreatic stone protein, and the Fc epsilon receptor for immunoglobulin E. Analysis of the binding properties of IX/X-bp revealed that it binds to the gamma-carboxyglutamic acid (Gla)-containing domains of factors IX and X [Atoda, H. et al. (1994) Eur. J. Biochem. 224, 703-708]. In the present study, we isolated another anticoagulant protein that binds to factor IX but is not to factor X. This protein, designated IX-bp, inhibited factor IXa-induced clotting but not factor Xa-induced clotting, whereas IX/X-bp inhibits both. The concentration of IX-bp for half-maximal binding to solid-phase bovine factor IX was 0.4 nM whereas IX-bp did not bind to factor X even at 40 nM. The binding of IX-bp to solid-phase factor IX was inhibited by the addition of Gla-domain peptide of factor IX, indicating that IX-bp binds to the Gla-domain region of factor IX. IX-bp had two Ca(2+)-binding sites with different affinities for Ca2+ ions. At pH 7.5, the apparent Kd values for these sites were 14 and 130 microM, respectively. IX-bp was a two-chain protein (27.5-kDa band before reduction and 16.8- and 15.7-kDa bands after reduction on SDS-PAGE) and it reacted with immunoglobulin G against IX/X-bp. The complete amino acid sequence of IX-bp was determined. The 16.8-kDa chain (A chain) of IX-bp consisted of 129 residues, of which 19 were different from those in the A chain of IX/X-bp (129 residues). The sequence of the 15.7-kDa chain (B chain) was identical to that of the B chain of IX/X-bp (123 residues). We conclude that IX-bp is a protein that is structurally similar to but functionally different from IX/X-bp. The difference of binding specificity between IX-bp and IX/X-bp presumably arises from the sequence differences in the A chains.
authors: Department of Biochemistry, Meiji College of Pharmacy, Tanashi, Tokyo.
authors: H
authors: H
authors: Atoda
authors: M
authors: M
authors: Ishikawa
authors: E
authors: E
authors: Yoshihara
authors: F
authors: F
authors: Sekiya
authors: T
authors: T
authors: Morita
mesh_terms: D000595:Amino Acid Sequence; D005591:Chemical Fractionation; D003435:Crotalid Venoms / Q000737:chemistry*; D005170:Factor X / Q000737:chemistry; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D003435:Crotalid Venoms; D011956:Receptors, Cell Surface; C029748:Trimeresurus venoms; C052276:factor IX-IXa receptor; D005170:Factor X