Structural and functional characterization of platelet receptor-mediated factor VIII binding

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.     

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.     

Replacing the Factor VIII C1 Domain with a Second C2 Domain Reduces Factor VIII Stability and Affinity for Factor IXa

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIII_C2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (∼11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIII_C2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (∼2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIII_C2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIII_C2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (∼10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIII_C2C2 variant (∼4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIII_C2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.     

The factor IXa second epidermal growth factor (EGF2) domain mediates platelet binding and assembly of the factor X activating complex.

Previously we have determined that residues 88-109 (but not Arg(94)) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725-5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), and FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K(d) values in 10(-9) m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (1.1 +/- 0.09, 5.0 +/- 0.90). These K(d) values are in good agreement with K((d)(app)) values (in 10(-9) m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loop3) (0.7 +/- 0.05, 3.0 +/- 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V(max) of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu(84)-Val(128)) was an effective inhibitor of FIXa binding to platelets in both the presence (K(i) = 0.7 x 10(-6) m) and the absence (K(i) = 1.5 x 10(-6) m) of FVIIIa and FX. We conclude that residues 88-109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex.ut not Ar     

Backbone resonance assignments of the C2 domain of coagulation factor VIII

Factor VIII (FVIII, other clotting factors are named similarly) is a glycoprotein that circulates in the plasma bound to von Willebrand factor. During the blood coagulation cascade, activated FVIII (FVIIIa) binds to FIXa and activates FX in the presence of calcium ions and phospholipid membranes. The C1 and C2 domains mediate membrane binding that is essential for activation of the FVIIIa–FIXa complex. Here, ¹H, ¹³C, and ¹⁵N backbone chemical shift assignments are reported for the C2 domain of FVIII, including assignments for the residues in solvent-exposed loops. The NMR resonance assignments, along with further structural studies of membrane-bound FVIII, will advance understanding of blood-clotting protein interactions.     

Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase

Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.     

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

Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys88-Cys99; loop 2, Cys95-Cys109) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of gamma-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of Km(app) and Kd(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (Kd(app)FIXa approximately 1.1 nm, Vmax approximately 12 nm min(-1)), N89A displayed an increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I90A had an increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V107A had an increase of approximately 3-fold in Kd(app)FIXa and approximately 4-fold decrease in Vmax. We conclude that residues Asn89, Ile90, and Val107 within loops 1 and 2 (Cys88-Cys109) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.     

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: A computational protein–protein docking and molecular dynamics refinement study

Coagulation factor X (FX) zymogen activation by factor IXa (FIXa) enzyme plays a critical role in the middle-phase of coagulation cascade. The activation process is catalytically inert and requires FIXa binding and complex formation with co-factor VIIIa (FVIIIa). In order to understand the structural details of the FVIIIa:FIXa complex, we employed knowledge-driven protein–protein docking and aqueous-phase MD refinement methods to develop a stable structural complex between FVIIIa and FIXa. The model shows that all four domains of FIXa wrap across FVIIIa that spans the co-factor binding surface of A2, A3 and C1 domains. The region surrounding the 558-helix of the A2-domain of FVIIIa is predicted to be the key interaction site with the helical segments of Lys293–Lys301 and Asp332–Arg338 residues of the serine-protease domain of FIXa. The hydrophobic helical stack between the GLA and EGF1 domains of FIXa is predicted to be primary interacting region with the A3–C2 domain interface of FVIIIa.     

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803–1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803–1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803–1810 and 1811–1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811–1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811–1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803–1810 and FVIII/FV 1811–1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811–1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811–1818 contributes to FIXa binding. Both regions 1803–1810 and 1811–1818 contribute to FVIIIa stability.     

Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding

During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased k(cat) dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXa(Y225P), a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold reduced k(cat). Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12-fold, implicating its role in physiologic coagulation.     

The interface between the EGF2 domain and the protease domain in blood coagulation factor IX contributes to factor VIII binding and factor X activation

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.     

Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase

Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.     

Factor VIII Lacking the C2 Domain Retains Cofactor Activity in Vitro

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated ΔC2) to characterize the contributions of the C1 domain to function. Binding affinity of the ΔC2 variant to phospholipid vesicles as measured by energy transfer was reduced ∼14-fold. However, the activity of ΔC2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions (∼4-fold) were observed in the functional affinity of ΔC2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability (∼3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as ∼60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function.     

Residues 88-109 of factor IXa are important for assembly of the factor X activating complex.

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)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127), and point mutants (FIXaR94D and FIXa(loop1)G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced V(max) of FX activation (nm FXa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were normal. For all of the FIXa proteins, K(m)((app)) values were normal as were EC(50) values for interactions with FVIIIa. However, K(d)((app)) (in nm) for the FX activating complex assembled on phospholipid vesicles was increased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and FIXaR94D (2.2 +/- 0.09) suggesting that reduced V(max) is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg(94)) are important for normal assembly of the FX activating complex on phospholipid vesicles.     

Factor VIII A3 domain residues 1793–1795 represent a factor IXa-interactive site in the tenase complex

BackgroundFactor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811–1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790–1798 form a V-shaped loop, and juxtapose residues 1811–1818 on the extended surface of FVIIIa.AimTo examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790–1798.Methods and resultsSpecific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790–1798 and 1811–1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC₅₀; 19.2 and 42.9 μM, respectively), in keeping with a possible role for the 1790–1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5–2.2-fold greater K_D compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the K_m by 1.6–2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the K_m was increased by 3.4-fold and the V_max was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction.ConclusionThe 1790–1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.     

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.     

A catalytic domain exosite (Cys527-Cys542) in factor XIa mediates binding to a site on activated platelets

The zymogen, factor XI, and the enzyme, factor XIa, interact specifically with functional receptors on the surface of activated platelets. These studies were initiated to identify the molecular subdomain within factor XIa that binds to activated platelets. Both factor XIa (Ki approximately 1.4 nM) and a chimeric factor XIa containing the Apple 3 domain of prekallikrein (Ki approximately 2.7 nM) competed with [125I]factor XIa for binding sites on activated platelets, suggesting that the factor XIa binding site for platelets is not located in the Apple 3 domain which mediates factor XI binding to platelets. The recombinant catalytic domain (Ile370-Val607) inhibited the binding of [125I]factor XIa to the platelets (Ki approximately 3.5 nM), whereas the recombinant factor XI heavy chain did not, demonstrating that the platelet binding site is located in the light chain of factor XIa. A conformationally constrained cyclic peptide (Cys527-Cys542) containing a high-affinity (KD approximately 86 nM) heparin-binding site within the catalytic domain of factor XIa also displaced [125I]factor XIa from the surface of activated platelets (Ki approximately 5.8 nM), whereas a scrambled peptide of identical composition was without effect, suggesting that the binding site in factor XIa that interacts with the platelet surface resides in the catalytic domain near the heparin binding site of factor XIa. These data support the conclusion that a conformational transition accompanies conversion of factor XI to factor XIa that conceals the Apple 3 domain factor XI (zymogen) platelet binding site and exposes the factor XIa (enzyme) platelet binding site within the catalytic domain possibly comprising residues Cys527-Cys542.     

Down-regulation of factor IXa in the factor Xase complex by protein Z-dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a serpin inhibitor of coagulation factor (F) Xa dependent on protein Z, Ca2+, and phospholipids. In new studies, ZPI inhibited FIXa in the FXase complex. Since this observation could merely represent inhibition of the FXa product whose activity was measured, inhibition of FIXa was investigated five ways. 1) FXase incubation mixtures with/without ZPI/protein Z were diluted in EDTA; FXa activity was measured after reversal of its inhibition. 2) FXase incubation mixtures were immunoblotted for FXa product. 3) FX activation peptide region was 3H-labeled; release of 3H was used to measure FXase activity. 4) Activity was monitored in a FIXa-based clotting assay. 5) FIXa amidolytic activity was measured. In all cases, FIXa was inhibited by subphysiologic levels of ZPI. Unlike inhibition of FXa, inhibition of FIXa did not strictly require protein Z. Low concentrations of FVIIIa increased the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure. Unusual time courses were observed for inhibition of both FIXa in the FXase complex and FXa in the prothrombinase complex. Activity loss stabilized in <100 s at a level dependent on ZPI concentration, suggesting equilibrium interactions rather than typical covalent serpin-protease interactions. Surface plasmon resonance binding experiments revealed binding and dissociation of ZPI/FIXa with Kd (app) of 9-12 nm, similar to the concentration of ZPI needed for 50% inhibition. ZPI may be an unusual physiologic regulator of both the intrinsic FXase and the prothrombinase complexes.     

Factor VIIIa A2 Subunit Shows a High Affinity Interaction with Factor IXa: CONTRIBUTION OF A2 SUBUNIT RESIDUES 707-714 TO THE INTERACTION WITH FACTOR IXa*

Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (K_d = 3.9 ± 1.6 nm) that was similar to the A3C1C2 subunit (K_d = 3.6 ± 0.6 nm). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. V_max values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent K_d relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys⁷⁰⁷, Asp⁷¹², and Lys⁷¹³ demonstrated the greatest increases in apparent K_d (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.     

The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.