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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.
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