Residues 88–109 of Factor IXa Are Important for Assembly of the Factor X Activating Complex

Abstract

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF) FIXa is a serine protease that participates in the intrinsic pathway of blood coagulation. FIXa activates FX as part of the intrinsic FX activating complex. The FX activating complex consists of FIXa, FVIIIa (a nonenzymatic cofactor), and FX (the normal macromolecular substrate) assembled on a procoagulant surface (8, 9). Under physiological conditions, thrombinactivated platelets or endothelial cells can provide this surface (10 -13). Additionally, synthetic phospholipid vesicles containing phosphatidylserine can also support assembly of the FX activating complex and activation of FX (8, 14). Assembly of the surface-bound FX activating complex results in a dramatic increase (ϳ20 million-fold) in the catalytic efficiency (k cat /K m ) of FX activation versus that of FIXa alone in solution (9, 15). The surface localization of FIXa is a requisite step in the catalytic enhancement of FIXa when assembled in the FX activating complex (9, 14).-Recent investigations have focused on the contributions of the EGF-like domains to FIXa biochemistry. Analysis of patient data suggests that residues contained within EGF1 and EGF2 are important for FIX(a) functions. Several point mutations within the EGF-like domains result in dysfunctional FIX(a) activity and a bleeding tendency (3,16). Investigations of the EGF-like domains have found several important functions for these domains. EGF1 is important for interactions with FVIIa/TF (17), FVIIIa (18), and FX (19,20). EGF1 does not appear to be important for binding to surfaces such as phospholipids (21), platelets, (22), or endothelial cells (23). While EGF2 has not been extensively characterized, experiments have indicated its importance for activation of FX. A chimeric FIXa protein in which both EGF1 and EGF2 domains were replaced by those of FX possessed about 4% clotting activity (24). In contrast, a chimeric protein with the FX EGF1 domain but the wild type FIX EGF2 domain was entirely normal (24), suggesting an essential function for EGF2. More recent experiments from our laboratory have indicated that EGF2 is likely involved in mediating surface binding to both platelets and phospholipids (25,26).To further define the contribution of the EGF2 domain to surface binding, we have prepared several chimeric FIXa proteins (see Fig. 1

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

“…We also noted that clotting was not affected by insertion of the R94D mutation. Our results are in line with those demonstrating that R94D is not important for assembly of the Xase complex on phospholipid vesicles (41). We believe that the results from our experiments show that the Glu 78 -Arg 94 salt bridge is not essential for maintenance of the FIX structure but that other interactions in this area are sufficient to maintain proper alignment of the light chain domains.…”

Identification of Residues Asn89, Ile90, and Val107 of the Factor IXa Second Epidermal Growth Factor Domain That Are Essential for the Assembly of the Factor X-activating Complex on Activated Platelets

“…1) to identify "candidate" residues that are highly conserved among species and different from those in FVII, because residues in FVII molecules have been shown to be ineffective in mediating platelet surface-mediated FX activation complex assembly (2,6). Recombinant FIXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared to study the contributions of these residues to FX activation complex assembly on activated platelets.…”

“…However, this contribution is considered to be structural rather than direct, because the segment FIXa variants showed normal FVIIIa binding in surface plasmon resonance experiments (36). Kinetic studies using recombinant FIXa chimeras have consistently shown that residues in the EGF2 domain do not mediate FVIIIa association suggested by both normal kinetic stimulation by FVIIIa and normal K d(app)FVIIIa values (2)(3)(4)6). Thus, the contribution of the non-catalytic domain of FIXa to its interaction with FVIIIa appears to be mainly structural, i.e.…”

“…Previous studies from our laboratory have demonstrated that the zymogen, FIX, binds to a discrete number (n ϳ 250 sites per platelet) of receptors on the surface of activated platelets (K d ϳ 2.5 nM) that can also be occupied by the enzyme, FIXa, and are mediated by residues Gly 4 -Gln 11 within the Gla domain (2)(3)(4)(5)(6). In addition, FIXa, but not FIX, can bind to a site (n ϳ 250 sites per platelet, K d ϳ 0.5 nM) on activated platelets, mediated by residues 88 -109 (disulfide constrained loops 1 and 2) but not by residues 110 -124 (loop 3) within the EGF2 domain (2)(3)(4)(5)(6).…”

“…In addition, FIXa, but not FIX, can bind to a site (n ϳ 250 sites per platelet, K d ϳ 0.5 nM) on activated platelets, mediated by residues 88 -109 (disulfide constrained loops 1 and 2) but not by residues 110 -124 (loop 3) within the EGF2 domain (2)(3)(4)(5)(6). Occupancy of these binding sites is closely correlated with optimal rate enhancements of FX activation (Ͼ2 ϫ 10 8 -fold) in the presence of FVIIIa, emphasizing the physiological significance of platelet-receptor-mediated coagulation complex assembly (2,6,7). Recently, however, alanine-scanning mutagenesis studies of residues within the EGF2 domain of FIX have identified residues Asn 89 -Gly 93 that were implicated as critical for binding of FVIIIa (8).…”