The A1 and A2 Subunits of Factor VIIIa Synergistically Stimulate Factor IXa Catalytic Activity

Fay, Philip J.; Koshibu, Kyoko; Mastri, Maria

doi:10.1074/jbc.274.22.15401

Cited by 36 publications

(36 citation statements)

References 33 publications

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“…11 for a review). Recent studies have shown that modulation of factor IXa by the isolated A2 subunit enhances the k cat for factor Xa activation by ϳ100-fold (12) and that the isolated A1 subunit synergizes this effect (13). This A1-dependent increase in A2 cofactor activity (Ͼ15-fold) did not result from direct interaction with factor IXa but rather altered the interaction of A2 subunit with the protease.…”

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confidence: 97%

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Cofactor Activities of Factor VIIIa and A2 Subunit following Cleavage of A1 Subunit at Arg336

Rosenblum

Schmidt

Freas

et al. 2002

Journal of Biological Chemistry

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Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K d ‫؍‬ 1.8 M). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg 336 by activated protein C or factor IXa is inactivating. A truncated A1 (A1 336 ) showed similar affinity for A2 subunit (K d ‫؍‬ 0.9 M) and stimulated its cofactor activity to ϳ50% that observed for native A1. However, A1 336 was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1336 . Fluorescence energy transfer demonstrated that, although Fl-A1 336 /A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.Factor VIII is a protein cofactor for the serine protease factor IXa, which catalyzes the conversion of factor X to Xa. Factor VIII is synthesized as a multidomainal single-chained molecule (A1-A2-B-A3-C1-C2) (1), with a molecular mass of ϳ300 kDa (2, 3). Factor VIII circulates as a partial proteolyzed protein containing a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains), which are held together by a metal iondependent linkage. This procofactor is activated by thrombin, converting the dimer into a trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits whereas a weak affinity electrostatic interaction exists between A1 and A2 (5, 6). Factor VIIIa is considerably unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (5-7).Under physiological conditions, the K d for this interaction is ϳ260 nM (8, 9), however, under slightly acidic, pH and lower ionic strength, A2 and A1/A3C1C2 can be reconstituted to form the trimer with a K d of ϳ30 nM (8).The role of factor VIIIa is to bind factor IXa, generating the phospholipid-dependent intrinsic factor Xase complex, which increases the k cat for factor Xa formation by several orders of magnitude compared with factor IXa alone (10). At least two interactive sites have been identified for the enzyme-cofactor interaction. A high affinity, surface proximal site is likely formed by residues contained in the A3 domain and light chain of factors VIIIa and IXa, respectively, whereas a weaker affinity interaction involves residues localized to the A...

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“…lier studies, the isolated A1 and A2 subunits of factor VIIIa were prepared following proteolysis of recombinant factor VIII by thrombin (12,13). However, subsequent subunit purification yielded relatively low levels of A2 subunit due to continued association with the A1/A3-C1-C2 dimer.…”

Section: Isolation and Purification Of Factor Viiia Subunits-in Ear-mentioning

confidence: 99%

Cofactor Activities of Factor VIIIa and A2 Subunit following Cleavage of A1 Subunit at Arg336

Rosenblum

Schmidt

Freas

et al. 2002

Journal of Biological Chemistry

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“…Because this Asn is contained within the N-X-(T/S) N-linked glycosylation site consensus sequence (31), failure to identify this residue provides direct support for utilization of Asn 41 for N-linked glycosylation. Stimulation of A2 by A1 Forms-We previously demonstrated that the isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X (14), and this activity is markedly enhanced in the presence of A1 subunit (15,16). To gain insights into critical regions of A1 required for this stimulation of A2, the cofactor activity of A2 in the presence of the native form and the two truncated A1 forms were compared using a factor Xa generation assay.…”

Section: A1 and A1mentioning

confidence: 99%

“…Interactive sites for factor IXa are localized to A2 and A3 domains (11)(12)(13). Recent studies have shown that modulation of factor IXa by the isolated A2 subunit enhances the k cat for factor Xa activation by ϳ100-fold (14) and that the isolated A1 subunit synergizes this effect (Ͼ15-fold) to alter the interaction of A2 subunit with the protease (15,16).…”

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Altered Interactions between the A1 and A2 Subunits of Factor VIIIa following Cleavage of A1 Subunit by Factor Xa

Nogami¹,

Wakabayashi²,

Schmidt³

et al. 2003

Journal of Biological Chemistry

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Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1 336 ) was previously shown to possess similar affinity for A2 and retain ϳ60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys 36 that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1 37-336 ) showed little if any affinity for A2 (K d >2 M), whereas factor VIIIa reconstituted with A2 plus A1 37-336 /A3-C1-C2 dimer demonstrated significant cofactor activity (ϳ30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1 37-336 showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K m for factor X when A1 was cleaved at Arg 336 . These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K m for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasmabased assays.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is deficient or defective in individuals with hemophilia A. Factor VIII functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa. Factor VIII is synthesized as a multi-domain, single chain molecule (A1-A2-B-A3-C1-C2) (1) with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1-A2-B domains and a light chain consisting of the A3-C1-C2 domains. This procofactor is activated by cleavage at Arg 372 , Arg 740 , and Arg 1689 by thrombin and factor Xa, converting the dimer into the factor VIIIa trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits, whereas A2 is associated with a weak affinity by electrostatic interactions (5, 6). Factor VIIIa is unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (5-7). Under physiological conditions, the K d for this interaction is ϳ260 nM (8, 9); however, at slightly acidic pH and low ionic strength, this interaction is facilitated by an ϳ10-fold increase in the affinity (K d ϭ ϳ30 nM) (8).The role of factor VIIIa in the intrinsic factor Xase is to bind factor IXa, which increases the k cat for factor Xa formation by several orders of magnitude compared with factor IXa alone (10). Interactive sites for factor IXa are localized to A2 and A3 domains (11-13). Recent studies have shown that modulation of factor IXa by the isolated A2...

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“…In contrast, the factor VIIIa A2 domain directly modulates the catalytic activity of factor IXa, which is further enhanced by the A1 domain, markedly increasing the k cat for factor X activation. 14,15 Although the isolated A2 domain binds with low affinity to factor IXa, it contributes significantly to protease-cofactor affinity in the membrane-bound enzyme complex. 16 Thus, the factor IXa-A2 domain interaction appears to be the critical protein-protein interface for cofactor enhancement of factor X activation.…”

Section: Introductionmentioning

confidence: 99%

The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis

Buyue

Whinna

Sheehan

2008

Blood

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The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the proteasefactor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4-to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM of factor IXa increased thrombin generation to approach TF-stimulated thrombin generation at 100% factor IX. Factor IXa R170A demonstrated a 2-to 3-fold increase in peak thrombin concentration and 5-fold increase in velocity index, whereas the response for factor IXa R233A was blunted and delayed relative to wild-type protease. In hemophilia B mice, factor IX replacement reduced the average time to hemostasis after saphenous vein incision, and the time to occlusion after FeCl 3 -induced saphenous vein injury. At 5% factor IX, the times to occlusion for factor IX wild-type, R170A, and R233A were 15.7 minutes, 9.1 minutes (P < . IntroductionThrombin is the penultimate product of the coagulation cascade, generated in an explosive burst on stimulation of plasma with limiting concentrations of tissue factor. 1,2 The measurement of plasma thrombin generation has merits as a global test of coagulation, and enhanced levels of thrombin generation have been associated with increased risk of recurrent venous thrombosis. 3 Thus, the rate and magnitude of thrombin generation may be predictive of the coagulation phenotype of patients. 4,5 In vitro and ex vivo modeling of the coagulation cascade indicates that factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa) is the rate-limiting step for thrombin generation. 1,2,6 Intrinsic tenase activity is unstable because of the diffusional loss of the noncovalently bound factor VIIIa A2 domain. 7,8 The instability of this enzyme complex is presumed to be an important regulator of the coagulation response.The mechanism(s) for activation of factor IXa within the intrinsic tenase complex are poorly understood. Factor VIII circulates as a heterodimer of A1-A2-B and A3-C1-C2 peptides with domain structure and metal-binding functions similar to ceruloplasmin. 9 Factor VIII undergoes proteolytic activation by thrombin, resulting in an unstable, metal-dependent A1/A2/A3-C1-C2 heterotrimer with cofactor activity. 10,11 Factor VIII or factor VIIIa light chain (A3-C1-C2 domains) bind to factor IXa on the phospholipid surface with an affinity that approaches intact factor VIIIa but lack cofactor activity. 12,13 The isolated factor VIIIa A1 domain also lacks cofactor activity. In contrast, the factor VIIIa A2 domain directly modulates the catalytic activity of factor IXa, which is further enhanced by the A1 domain, markedly increasing the k cat for factor X activation. 14,15 Although the isolated A2 domain binds with low affinity to factor IXa, it contributes significantly to protease-c...

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The A1 and A2 Subunits of Factor VIIIa Synergistically Stimulate Factor IXa Catalytic Activity

Cited by 36 publications

References 33 publications

Cofactor Activities of Factor VIIIa and A2 Subunit following Cleavage of A1 Subunit at Arg336

Cofactor Activities of Factor VIIIa and A2 Subunit following Cleavage of A1 Subunit at Arg336

Altered Interactions between the A1 and A2 Subunits of Factor VIIIa following Cleavage of A1 Subunit by Factor Xa

The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis

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