Interaction of Factor IXa with Factor VIIIa

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The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca 2؉ and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558 -565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558 -565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa R333Q and by a helix replacement mutant (IXa helixVII in which helix 330 -338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa PCEGF1 in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K d(A2) values are: dEGR-IXa WT or dEGR-IXa PCEGF1 ϳ100 nM, dEGR-IXa R333Q ϳ1.8 M, and dEGR-IXa helixVII >10 M. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558 -565 of the A2 subunit. Apparent K d(peptide) values are: dEGR-IXa WT or dEGRIXa PCEGF1 ϳ4 M, and dEGR-IXa R333Q ϳ62 M. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558 -565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558 -565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.Physiologic blood clotting begins by exposure of blood to tissue factor (TF) 1 at an injury site and formation of the complex between TF and plasma factor VIIa. The TF⅐VIIa complex formed activates both factors IX and X (1, 2). Factor IXa thus generated forms a stoichiometric complex with factor VIIIa and also activates factor X in the presence of Ca 2ϩ and a suitable phospholipid (PL) surface (1, 2). The role of factor VIIIa in this complex is to increase the k cat by several orders of magnitude while PL primarily reduces the K m for the substrate factor X.Human factor IX circulates in blood as a single chain protein of 415 amino acids. Upon activation (by factor XIa/Ca 2ϩ or TF⅐VIIa/Ca 2ϩ

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Factor IXa:Factor VIIIa Interaction

Bajaj

Schmidt

Mathur

et al. 2001

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“…The impact of the FX mutations on activation by TF⅐FVIIa was, however, Clearly, our results suggest that the potential exosite-1 of FX is nonessential for its activation by RVV-X, TF⅐FVIIa, or FVIIIa⅐FIXa. The contribution of loops 34 -40 and 70 -80 of FIX to its activation by TF⅐FVIIa has not been studied in detail, but its calcium-binding site (within segment 70 -80) is not involved in the reaction (35). In contrast, positive charges within proexosite-1 of prothrombin are essential for its activation by prothrombinase (36,37), whereas E39K prothrombin is normally activated (17).…”

Section: Plasma (Ex Vivo) Studies Of Fxa and Derivatives-mentioning

confidence: 99%

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

Bianchini

Pike

Bonniec

2004

Coagulation factor X (FX) 1 is a vitamin K-dependent zymogen activated into FXa by cleavage at a single bond (Arg 15 -Ile 16 in the chymotrypsin numbering system 2 ) hydrolyzed by either of two complexes: FVIIa bound to tissue factor (TF), which triggers coagulation after injury, or FIXa associated with FVIIIa, which amplifies thrombin production after initiation of coagulation. FXa is the only endogenous prothrombin activator, but substantial thrombin production occurs only within the prothrombinase complex, where FXa binds to FVa in the presence of calcium on an appropriate phospholipid surface. Two inhibitors control FXa activity, viz. the tissue factor pathway inhibitor (TFPI) and antithrombin.Extended binding sites (exosites) play a crucial role in virtually any substrate, cofactor, or inhibitor recognition within the coagulation cascade. Perhaps best characterized is the case of thrombin taking advantage of two exosites for extended interactions in numerous functions (1). Exosite-1 is formed by a set of basic residues comprising loops 34 -40 and 70 -80, which neighbor each other in the folded structure (2); it interacts with fibrinogen, thrombomodulin, platelet activator receptor-1, FV, FVa, heparin cofactor II, and hirudin. Exosite-2, comprising loop 91-102 and helices 165-173 and 233-245 (3), is also formed by a set of basic residues that interact with heparin, with the chondroitin sulfate of thrombomodulin, and within prothrombin with kringle-2 (4). In FVII, the region corresponding to exosite-1 of thrombin is critical for FX activation (5, 6); in FIXa, this region is also important for FX activation in the absence of FVIII and for its inhibition by antithrombin in the absence of heparin (7). In contrast to thrombin, FVIIa, FIXa, and FXa share a negatively charged patch within segment 70 -80, whereas FXa is unique in that both loops 34 -40 and 70 -80 are negatively charged. Thus, the region of FXa corresponding to exosite-1 of thrombin forms a negative cluster, raising the question as to whether it could constitute a functional exosite. FXa has a unique macromolecule substrate, but exosites may also participate in its inhibition and/or in the activation of its zymogen. In fact, a number of studies have established that exosite(s) must be critical in several FXa functions. Electrostatic interactions involving loop 34 -40 of FXa appear to play a significant role in binding to the second Kunitz domain of TFPI (8,9). FXa also appears to interact with a complementary surface on pentasaccharide-activated antithrombin (10), and the region of FXa topologically equivalent to exosite-2 of thrombin seems to be involved in the binding of heparin and FVa (11). Above all, prothrombin recognition by prothrombinase undoubtedly involves one or more exosites on .In a previous study (15), we reported that the catalytic groove of FXa is minimally selective with unconstrained peptides and that FVa has little effect, if any, on this selectivity. These two observations therefore also favor the hypothesis that FXa uses seconda...

“…When the locations of these three essential amino acids were displayed using the human FIXa crystal structure (PDB: 1RFN, not shown) they form a planar surface, which the present studies suggest forms a site utilized for binding to activated platelets, consisting of two hydrophobic residues (Ile and Val) and an uncharged polar residue (Asn) that can participate in hydrogen bonding. Extensive studies have been carried out to identify regions in FIXa that are essential in surface (14 -21) or cofactor interaction (1,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Regions 301-303 and 333-339 in the FIXa catalytic domain are implicated as FVIIIa-interactive sites, because mutations in these regions in recombinant FIXa chimeric proteins had deleterious effects on FX activation with significantly increased EC 50VIIIa values (26), results supported by direct equilibrium binding studies (32).…”

Section: Fixa Plateletsmentioning

confidence: 99%

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

Yang

Chang

Lin

et al. 2004

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, Cys 88 -Cys 99 ; loop 2, Cys 95 -Cys 109 ) 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 ␥-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 K m(app) and K d(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 (K d(app)FIXa ϳ 1.1 nM, V max ϳ 12 nM min ؊1 ), N89A displayed an increase of ϳ20-fold in K d(app)FIXa and a decrease of ϳ20-fold in V max ; I90A had an increase of ϳ5-fold in K d(app)FIXa and ϳ10-fold decrease in V max ; and V107A had an increase of ϳ3-fold in K d(app)FIXa and ϳ4-fold decrease in V max . We conclude that residues Asn 89 , Ile 90 , and Val 107 within loops 1 and 2 (Cys 88 -Cys 109 ) 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.