The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

Abstract

The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu 84 -Thr 87 and the factor IX-specific loop Asn 89 -Lys 91 were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn 89 -Lys 91 resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu 84 -Thr 87 between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa. Factor IX (FIX)1 is a vitamin K-dependent serine protease precursor that participates in the process of blood coagulation (1, 2). Factor IX is converted into an active serine protease upon cleavage by either factor XIa (FXIa) or the factor VIIa (FVIIa)-tissue factor complex (3, 4). Proteolysis occurs at restricted cleavage sites and results in the formation of the two-chain enzyme factor IXa (FIXa). FIXa is composed of a light chain that contains an N-terminal Gla domain followed by a short hydrophobic region and two epidermal growth factor (EGF)-like domains (5). The heavy chain comprises the protease domain with the catalytic center. FIXa shares its typical domain structure with related serine proteases of the blood coagulation cascade, including FVIIa, factor Xa (FXa), and activated protein C (6, 7). All of these enzymes contain two EGF-like domains that display considerable sequence homology with the EGF-like modules in factor IXa (8), indicating that these domains are similar in structure and function. Indeed, FIXa, FXa, and FVIIa are similar in that calcium binding to the first EGF-like module is essential for conserving the linkage between its N-terminal half and the N-terminal Gla domain in these proteins (for review, see Ref. 9). However, with respect to the relative orientations of the two EGF-like modules, the three-dimensional crystal structures reveal that this is different in each of these proteases. The connection between these modules is flexible within the structures of F...

Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

“…Briefly, 25 pM of relipidated TF was incubated with 8 nM FVIIa for 5 min. FXa generation was started by the addition of 400 nM pd-FX followed by immediate addition of preformed TFPI-recombinant FXa derivatives (25,50, and 100 pM). After various incubation times, aliquots were removed, diluted in a stop solution containing EDTA (10 mM final), and assayed for FXa formation using the synthetic substrate S-2765 (500 M).…”

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

“…For instance, the interaction of membrane-bound FVIIIa with FIXa to form the Xase complex increases the V max for FX activation by several orders of magnitude (12). Three-dimensional structures of FIXa and molecular models of FVIIIa (based on its homology with ceruloplasmin) have allowed preliminary models of the FIXa-FVIIIa complex to be constructed (13,14). These models accommodate the well established facts that the Gla domain of FIXa and the C2 domain of FVIIIa interact with biological membranes.…”

Residues Phe342–Asn346 of Activated Coagulation Factor IX Contribute to the Interaction with Low Density Lipoprotein Receptor-related Protein

“…Recombinant FIX-Stable cell lines producing FIX variants were obtained by the calcium phosphate co-precipitation method and selection with geneticin (32). As reported previously, the expression system used in this study yields recombinant FIX molecules with normal calciumdependent properties and similar activities for recombinant wild type and plasma-derived FIXa (26,33,35) with an average Gla content of 11 mol of Gla/mol of protein (36). Recombinant FIX was purified by affinity chromatography employing anti-FIX monoclonal antibody CLB-FIX 14 from concentrated medium obtained by culturing cell lines producing FIX in 1-liter cell factories (35).…”