Factor IXa:Factor VIIIa Interaction

Self Cite

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8 Å were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55°C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is decreased or defective in individuals with hemophilia A. Factor VIII circulates as a non-covalent, metal ion-dependent heterodimer consisting of a heavy chain comprised of A1(a1)A2(a2)B 2 domains and a light chain comprised of (a3)A3C1C2 domains (see Ref. 1 for review). The factor VIII procofactor is activated by limited proteolysis catalyzed by thrombin or factor Xa to yield the cofactor, factor VIIIa, following cleavages at the a1A2, a2B, and a3A3 junctions. Thus, factor VIIIa is a heterotrimer of subunits designated A1, A2, and A3C1C2. Factor VIIIa functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (see Ref. 1 for review). The role of the cofactor in the intrinsic factor Xase complex is to increase the catalytic efficiency of this reaction by several orders of magnitude. Factor Xase is self-dampening as a result of instability in the factor VIIIa cofactor due to weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer (2, 3). Limited information is available regarding the association of the A2 subunit in factor VIIIa. Fluorescence energy transfer results (4) and activity assays following swapping the human A1 domain for the porcine homolog (5) suggest that interactions o...

Section: Discussionmentioning

confidence: 99%

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

Wakabayashi

Fay

2008

Self Cite

“…Based upon experimental data supporting a direct interaction of the 558-loop of A2 with the 330-helix of factor IXa, Bajaj et al (19) proposed a model for this interface between the protease domain and the A2 subunit following docking of these structures. In the model, Asp-560 is suggested to electrostatically interact with Arg-338 (Arg-170 using the chymotrypsin number system) of factor IXa.…”

Section: A2 Variantmentioning

confidence: 99%

“…One region in A2, the 558-loop was identified as a factor IXa-interactive site by both peptide inhibition studies (17) and by the capacity of factor IXa to selectively protect factor VIIIa from activated protein C-catalyzed cleavage at Arg-562 (18). More recently, Bajaj et al (19) suggested that residues within the A2 558-loop bind to the 330-helix in factor IXa and modeled this interaction. According to this model, electrostatic, hydrophobic interactions and hydrogen bonds contribute to forming this interface.…”

mentioning

confidence: 99%

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

Self Cite

Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cellexpressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K d for A2. The second assay determined the K d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K d and k cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.Factor VIIIa is an essential blood coagulation protein that acts as a cofactor for the serine protease factor IXa in the conversion of factor X to Xa during the propagation phase of coagulation. Defects or deficiencies in factors VIII and IX result in hemophilia A and B, respectively. Factor VIII is synthesized as a multidomain, single chain precursor with a molecular mass of ϳ300 kDa and domain structure A1-A2-B-A3-C1-C2. It circulates primarily as a heterodimer resulting from proteolysis at the B-A3 junction wherein the heavy chain (A1A2B) and light chain (A3C1C2) are associated by metal ion-dependent and independent linkages (see Ref. 1 for review). Factor VIII is activated by limited proteolysis catalyzed by thrombin or factor Xa to the active cofactor, factor VIIIa. These cleavages convert the heterodimer or single chain precursor to the heterotrimer (2, 3) with the heavy chain-derived A1 subunit and light chain-derived A3C1C2 subunit maintaining stable association, whereas the A2 subunit is weakly associated with the A1/A3C1C2 dimer through electrostatic interactions (4, 5). Factor VIIIa can be reconstituted from the isolated A2 subunit and A1/A3C1C2 dimer to regenerate high levels of cofactor activity (3, 5).Association of factors VIIIa and IXa on a phospholipid surface forms the intrinsic factor Xase complex, which increases the cataly...

“…Positioning of the Fab took into account only the overall shape complementarity between FIXa and the Fab structure; a full docking protocol could not be performed, since the amino acid sequence of mAb AW is not known. An approximate model for the FIXa-Fab complex was then positioned next to the FVIIIa model using information previously reported for FVIIIa, and further optimization of the system was performed interactively (13,32). Modeling and structural analysis were performed on Silicon Graphics O 2 and FUEL workstations (Mountain View, CA) with the Accelrys package (available on the World Wide Web at www.accelrys.com; Accelrys, San Diego, CA).…”

Section: Effect Of Mab Aw On the Activation Of Fix By Fxia And Fviia⅐tf-mentioning

confidence: 99%

The N-terminal Epidermal Growth Factor-like Domain of Coagulation Factor IX

Persson¹,

Villoutreix

Thämlitz³

et al. 2002

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.