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The Distinct Roles That Gln-192 and Glu-217 of Factor Ix Play in Selectivity for Macromolecular Substrates and Inhibitors
No full textIn this paper, we report functional characterization of positions 192 and 217 (chymotrypsinogen numbering system) in human factor IX and discuss the distinction and similarity of these two sites among the blood coagulation factors. Recombinant factor IXQ192E (residue glutamine at position 192 replaced by glutamic acid), IXQ192K, IXE217D, and IXE217 R proteins exhibited 11%, 46%, 39%, and 2% of the wild-type factor IX's clotting activity, respectively. Binding of these variants to factor VIIIa (FVIIIa) was inefficient compared to that of wild-type factor IX, and the dissociation constants doubled for IXQ192E, 3-fold higher for IXQ192K and 4-fold higher for both IXE217D and IXE217R. In the presence of FVIIIa, all variant factor IX hydrolyzed factor X at the catalytic efficiencies correlating with respective clotting activities. However, FVIIIa greatly enhanced the catalytic efficiency of both IXE217 variants to a greater extent (similar to7 x 10(4)-fold) as compared to its effect on the wild- type factor IXa and the other two IXQ 192 variants [by a factor of (1-2) x 10(4)]. Moreover, while both IXQ192 variants demonstrated small substrate selectivity similar to that of wild-type factor IXa, the selectivity of both IXE217 variants was greatly altered. Mutations at position 192 disturbed the interaction of factor IXa with physiological inhibitors. Although all variants formed an SDS-stable complex with antithrombin III ( ATIII) equally well in the presence of heparin and were readily inhibited by ATIII in the absence of heparin, activated IXQ192K exhibited a slower stable complex formation with ATIII without heparin. On the other hand, only IXQ192E showed decreased interaction with TFPI. Our results demonstrate that positions 192 and 217 play different roles unique to factor IX in specifying the interaction of factor IX with substrates and inhibitors
Identification of Residues in Asn 89, Ile 90 and Val 107 of the Factor Ixa Second Epidermal Growth Factor Domain That Are Essential for the Assembly of the Factor-X Activating Complex on Activated Platelets
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Identification of residues Asn 89, Ile 90 and Val 107 of the factor IXa second epidermal growth factor domain that are essential for the assembly of the factor X activating complex on activated platelets
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Identification of functionally important residues of the epidermal growth factor-2 domain of factor IX by alanine-scanning mutagenesis - Residues Asn(89)-Gly(93) are critical for binding factor VIIIa
No full textThis paper describes the consequences of alanine-scanning
mutagenesis on 28 positions of the second epi-dermal
growth factor (EGF-2) domain of factor IX. We
identified four positions of Gln 97 , Phe 98 , Tyr 115 , and
Leu 117 that are critical for secretion of factor IX. Of the
remaining mutations, 4 mutants (V86A, E113A, K122A,
and S123A) are as active as wild-type factor IX (IXwt); 16
(D85A, K100A, N101A, D104A, N105A, R116A, E119A,
T87A, I90A, K91A, R94A, E96A, S102A, K106A, T112A, and
N120A) retain reduced but detectable activity, and 4
(N89A, N92A, G93A, and V107A) are nearly inert in the
clotting assay. Both factor XIa and the factor VIIa-tissue
factor complex effectively catalyzed the activation of
these mutants except N89A. The mutant V107A failed to
form the factor tenase complex with factor VIIIa be-cause
of a 35-fold increase in Kd . The mutants N89A and
N92A did not compete with factor IXwt for factor VIIIa
binding, and G93A exhibited a 6-fold increase in Ki val-ues
in the competitive binding assay. It appears that
mutations at these positions have significantly affected
the interaction between factor IX and factor VIIIa, al-though
other mutations had little effect on the binding
of factor IX to factor VIIIa. Mutations in two regions,
Thr 87 –Gly 93 and Asn 101 –Val 107 , significantly increased
the Km value of factor IXa (2–10-fold) in cleavage of fac-tor
X in the absence of factor VIIIa. In the presence of
factor VIIIa, the catalytic efficiency of each mutant to-ward
factor X paralleled its clotting activity. Briefly, we
propose two relatively distinctive functions of factor IX
for two adjacent regions in the EGF-2 domain; the first
loop region (residues 89–94) is involved with the binding
of its cofactor, factor VIIIa, and the third loop with
connected -sheets (residues 102–108) is involved in the
proper binding to the substrate, factor X
A Novel Mutation Identified by the Polymerase Chain Reaction (PCR) and the Single Strand Conformation Polymorphism (SSCP)
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Hemophilia a of Chinese Origin:Analysis by the Single-Strand Conformation Polymorphism (SSCP) and Direct Sequencing
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