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PubMed2002

The N-terminal epidermal growth factor-like domain of coagulation factor IX. Probing its functions in the activation of factor IX and factor X with a monoclonal antibody.

The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor-like (EGF) domains and a serine protease domain. In this study, the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined, and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by activated factor XI, but activation by activated factor FVII-tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent k(cat) for factor X both in the presence and absence of activated factor VIII. Based on these results, we produced a preliminary model of the structure of the activated factor IX-activated factor VIII-AW complex on the surface of phospholipid. The model suggests that in the Xase complex, EGF1 of activated factor IX is not involved in direct binding to activated factor VIII. Studies of the interaction of antibody AW with a mutated FIX molecule (R94D) also suggest that the Glu(78)-Arg(94) salt bridge is not important for maintaining the structure of FIX.

Amides↗
PubMed2005

An ordered sequential mechanism for Factor IX and Factor IXa binding to platelet receptors in the assembly of the Factor X-activating complex.

To define the contributions of the Omega-loop of the Gla (gamma-carboxyglutamic acid) domain and the EGF2 (second epidermal growth factor) domain of FIXa (Factor IXa) in the assembly of the FX-activating complex on activated platelets and phospholipid membranes, three recombinant FIXa chimeras were prepared with corresponding residues from the homologous coagulation protein, FVII: (i) Gly4-Gln11 (FIXa7Omegaloop), (ii) Cys88-Cys124 (FIXa7EGF2), and (iii) both Gly4-Gln11 and Cys88-Cys124 (FIXa7Omegaloop7EGF2). All three chimeras were similar to wild-type FIXa, as assessed by SDS/PAGE, active-site titration, content of Gla residues, activation rates by FXIa and rates of FXa generation in solution. Titrations of FX or FVIIIa on SFLLRN peptide-activated platelets and on phospholipid vesicles in the presence of FVIIIa revealed normal substrate and cofactor binding to all chimeras. In kinetic assays in the presence of phospholipid vesicles and FVIIIa, compared with wild-type FIXa K(d, app) approximately 4 nM, the FIX7Omegaloop chimera showed a 1.6-fold increase in K(d, app), the FIX7EGF2 chimera had a 7.4-fold increase in K(d, app), and the FIX7Omegaloop7EGF2 chimera showed a 21-fold increase in K(d, app). In kinetic assays and equilibrium platelet-binding assays with activated platelets and FVIIIa, compared with wild-type FIXa (V(max) approximately 5 nM min(-1); K(d, app) approximately 0.5 nM; B(max) approximately 550 sites/platelet; K(d) approximately 0.5 nM), the FIX7Omegaloop chimera displayed 2-fold decreases in V(max) and B(max) and 2-fold increases in K(d, app) and K(d). The FIX7EGF2 chimera displayed 2-fold decreases in V(max) and B(max) and 10-fold increases in K(d, app) and K(d). The FIX7Omegaloop7EGF2 chimera showed non-saturable curves and severely impaired rates of FXa generation, and non-saturable, non-specific, low-level binding to activated platelets. Thus both the Gla domain Omega-loop (Gly4-Gln11) and the EGF2 domain (Cys88-Cys124) are required to mediate the normal assembly of the FX-activating complex on activated platelets and on phospholipid membranes.

Amino Acid Sequence↗
PubMed1977

Factor IX antigen by radioimmunoassay. Abnormal factor IX protein in patients on warfarin therapy and with hemophilia B.

Factor IX, isolated from normal human plasma, was homogenous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. On the latter, it migrated as a single polypeptide chain with or without reducing agents and had an apparent mol wt of 62,000. After iodination by chloramine-T, a single peak of 125I was found on gels. Immunoelectrophoresis in agarose with rabbit antifactor IX sera gave a single arc against both isolated and partially purified factor IX preparations. The rabbit antibody was specific as it failed to inhibit the activities of prothrombin or factors VII or X in normal plasma. At an additional 20-fold dilution, factor IX activity was inhibited 50%. In a double-antibody radioimmunoassay, excess rabbit anti-human factor IX precipitated 90-95% of the 125I-human factor IX. Control without specific antibody gave 6-8%. Dilutions of a pool of normal human plasma paralleled dilutions of the isolated preparation and were used for the standard curve. Of 39 plasma samples from normal donors, the mean factor IX antigen level was 93% of that of a separate normal pool. The radioimmunoassay detected the abnormal factor IX produced in patients on warfarin therapy. After Al(OH)3 adsorption of warfarin treated patient's plasma, factor IX antigen, but not activity, was present in the supernate. Samples from 28 patients on warfarin gave a mean factor IX clotting activity of 27% with a mean antigen of 69%. The antigen level from the warfarin group was significantly lower than the antigen level of the normal group (P less than 0.001). The factor IX antigen level was then assessed in 36 patients from 29 pedigrees with hemophilia B. The median antigen level was 17% of normal. The distribution of the antigen level was wide with two patients around 100% of normal; only two had levels below the limits of resolution of the radioimmunoassay as currently performed (less than 2%). Within each of the five pedigrees in which more than one affected member was tested, activity and antigen levels were the same. The degree of neutralization of the antibody's inhibition of normal plasma by patient's plasma was highly correlated. Additional evidence for the detection of abnormal protein was provided by immunodiffusion of plasmas concentrated by lyophilization. Reactions of complete identity occurred between normal, a warfarin treated and a hemophilia B subject's plasmas.

Adolescent↗
PubMed1989

Factor IX Cardiff: a variant factor IX protein that shows abnormal activation is caused by an arginine to cysteine substitution at position 145.

Crude barium chloride eluates prepared from 12 unrelated patients with cross-reacting material positive (CRM+) haemophilia B were activated with celite eluate, the reaction products resolved after reduction by 13% SDS-PAGE, and factor IX antigenic material detected by probing with radiolabelled immunopurified rabbit anti-factor IX antiserum followed by autoradiography. Out of the 12, one sample showed faulty activation with the production of a stable reaction product with a MW compatible with that of a putative light chain-activation intermediate. In order to confirm this, two oligonucleotide primers that bracketed exon 6 of the factor IX gene were constructed and used to prime a polymerase chain reaction on DNA isolated from the patient's peripheral blood leucocytes. A single 489 nucleotide DNA fragment was obtained, gel purified, subcloned into M13, and DNA sequencing carried out on both strands. A single C to T transition was discovered that changed the Arg residue at position 145, the first residue of the first bond in the activation peptide, to a Cys, a result that confirmed the inferences drawn from the activation studies.

Antigens↗
PubMed2002

Role of the N-terminal EGF module of coagulation factor IX in activation of factors IX and X.

Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder haemophilia B. FIX contains a Gla module, two epidermal growth factor-like (EGF) modules, and a serine protease region. I characterized a monoclonal antibody and found that it recognizes an epitope around residues 72 and 80 in the C-terminal part of EGF1 in human FIX. The antibody exhibited 10-fold greater affinity for activated FIX (FIXa) than for the zymogen FIX, indicating the existence of intra-molecular communication between the serine protease region and EGF1. Binding of the antibody did not affect the amidolytic activity of FIXa, hence I could use the antibody during activation of FIX to show that the C-terminal part of EGF1 is of importance for the interaction with FXIa but not with FVIIa/TF. Considering activation of FX, it is a matter of debate whether EGF1 or FIXa interacts directly with FVIIIa. I activated FX in the presence and absence of the antibody and/or FVIIIa. The addition of antibody caused only a minor decrease in k(cat,app), and the major increase in k(cat,app) caused by the addition of FVIIIa occurred even in the presence of the antibody. This implies that EGF1 of FIXa is not directly involved in interaction with FVIIIa in the Xase complex. A model of the FIXa-FVIIIa complex, based on my findings and results from the literature, was constructed and indicated that EGF1 of FIXa does not interact directly with FVIIIa.

Amino Acid Sequence↗
PubMed1991

Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa.

We have previously used monoclonal antibodies to identify an epitope on the heavy chain of factor XIa that is a substrate-binding site for factor IX (Sinha, D., Seaman, F.S., and Walsh, P.N. (1987) Biochemistry 26, 3768-3775; Baglia, F.A., Sinha, D., and Walsh, P.N. (1989) Blood 74, 244-251). To define the factor XIa domain that binds factor IX, we have now screened a panel of factor XI heavy chain-derived synthetic peptides for their capacity to inhibit the formation of an activation peptide reflecting factor IX activation by factor XIa. Peptide Asn145-Ala176 (which is located in the second tandem repeat or A2 domain of the factor XI heavy chain) is a competitive inhibitor of factor IX activation by factor XIa with a Ki of 30 nM, whereas structurally similar peptides in the A1, A3, and A4 domains were required at 10-1000-fold higher concentrations for similar effects, and a synthetic peptide identical with a highly homologous region of the heavy chain A2 domain of prekallikrein (Tyr143-Ala176) had no effect on factor IX activation by factor XIa. Because detailed structural information is lacking, a potential three-dimensional structure for the factor XI A2 domain was calculated based on its sequence information in conjunction with previously determined structural constraints. The resulting structure depicted three juxtaposed beta-stranded stem-loops that, based on biological information, constitute a candidate surface for contact with factor IX. The A2 model was therefore used as a template in the rational design of three synthetic peptides (Ala134-Ile146 (peptide a), Leu148-Arg159 (peptide b), and Ile160-Leu172 (peptide c]. When peptides a and b or a and c were added together and the activation of factor IX by factor XIa was examined, a synergistic inhibitory effect was observed, compared with each peptide added individually, whereas peptides b and c showed additive effects. Our data suggest that the sequence of amino acids from Ala134 through Leu172 of the heavy chain of factor XI contains three antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of factor IX.

Amino Acid Sequence↗
PubMed1993

The metal-dependent conformational changes in factor IX associated with phospholipid binding. Studies using antibodies against a synthetic peptide and chemical modification of factor IX.

Factor IX undergoes two sequential metal-ion-dependent conformational transitions [Liebman, H.A. (1987) J. Biol. Chem. 262, 7605-7612]. The first transition is metal ion dependent, but cation nonselective. The second transition is metal ion selective for Ca(II) or Sr(II) and associated with the expression of conformational determinants necessary for phospholipid membrane binding. Using antibodies raised against a synthetic peptide containing Factor IX amino acid residues 28-46, it was demonstrated that the aromatic-amino-acid-stack domain (amino acid residues 41-46) of Factor IX is surface exposed in the metal-free conformer and internalized with the initial metal-ion-dependent conformational transition. The metal-ion-induced internalization of this region of Factor IX protects the tryptophan at position 42 from oxidation by N-bromosuccinimide. The oxidation of Factor IX tryptophan residues is associated with the rapid loss of coagulant activity, but protection of Trp42 allows for the continued expression of the Ca(II)-specific antigenic determinants on Factor IX and the retention of phospholipid binding. These results suggest that the Trp42 residue located in the aromatic amino acid stack domain of human Factor IX is internalized with the metal-ion-dependent conformational transition and is essential for the expression of the phospholipid membrane binding site on Factor IX.

Animals↗
PubMed1984

Monoclonal antibodies to factor IX: characterization and use in immunoassays for factor IX.

Monoclonal antibodies to factor IX have been characterized for potential use in purification of factor IX and for analysis of factor IX abnormalities seen in hemophilia B and other conditions. The properties of six monoclonal antibodies to factor IX are summarized in this report. Antibodies were selected for diverse reactions with factor IX and activated factor IX and which had been transferred to nitrocellulose. Two antibodies showed preference for the heavy chain of activated factor IX, one antibody showed preference for the intact factor IX molecule while three antibodies reacted with the heavy chain of activated factor IX and the intact factor IX molecule equally. Five monoclonal antibodies inhibited factor IX clotting activity. Two antibodies, most specific for factor IX by competitive binding assays, have proved to be useful in immunoassays for factor IX antigen in normal plasmas and in samples from hemophilia B patients.

Animals↗