Role of Factor VIII C2 Domain in Factor VIII Binding to Factor Xa

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We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC 50 ‫؍‬ 11 M) and partially blocked cleavage at the A2-B junction (IC 50 ‫؍‬ 100 M). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of ϳ50% and an activation rate constant of ϳ30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.Factor VIII, a plasma protein deficient of defective in individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa (1). Factor VIII is synthesized as a multi-domain single chain molecule (A1-A2-B-A3-C1-C2) consisting of 2332 amino acid residues with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B-domain linked to a light chain consisting of the A3, C1, and C2 domains (2-4).Factor VIII is converted into an active form, factor VIIIa, following limited proteolysis catalyzed by either thrombin or factor Xa (5). Cleavages at Arg 372 and Arg 740 of the heavy chain produce the 50-kDa A1 and 40-kDa A2 subunits. Cleavage of the 80-kDa light chain at Arg 1689 produces a 72-kDa A3-C1-C2 subunit. Additionally, cleavage by factor Xa at Arg 1721 produces a 67-kDa A3-C1-C2 subunit. Proteolysis at Arg 372 and Arg 1689 is essential for generating factor VIIIa cofactor activity (see for review see Ref. 6). Cleavage at the former site ...

Section: Discussionmentioning

confidence: 64%

Section: From the Departments Of ‡Biochemistry And Biophysics And ¶Mementioning

confidence: 99%

Section: Inhibition Of Factor Xa-catalyzed Factor VIII Activation By mentioning

confidence: 99%

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Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Nogami

Lapan

Zhou

et al. 2004

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“…FVIII binding to anhydro-thrombin in the current investigations was also not affected by the presence of vWF or PL (data not shown), suggesting that the thrombin-binding site in the C2 domain is separated from the vWF-and PL-binding sites. In our previous work (20) we identified the FXa-binding site within the C2 domain, and we demonstrated that binding of FXa was completely inhibited by the presence of the vWF. This is markedly in contrast to the binding profile of thrombin demonstrated in the current study.…”

Section: Competitive Inhibition Of Thrombin Proteolysis Of Lch Bymentioning

confidence: 99%

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Nogami¹,

Shima²,

Hosokawa³

et al. 2000

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Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg 1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Blood clotting factor VIII (FVIII) 1 is a crucial glycoprotein that accelerates the intrinsic coagulation cascade by acting as a cofactor of factor IXa in the tenase complex (1), and a deficiency of FVIII results in the common hereditary bleeding disorder, hemophilia A. FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (vWF) that stabilizes the synthesis and cofactor activity of FVIII (2-4). Mature FVIII is synthesized as a single chain polypeptide of approximately 300 kDa consisting of 2,332 amino acid residues with a mosaic domain structure arranged in the order of A1-A2-B-A3-C1-C2 (5-7) and secreted into plasma as variable series of heterodimers consisting of the heavy chain (HCh) and the light chain (LCh). The HCh is composed of the A1, A2, and parts of the B domain and exhibits size heterogeneity (90 -210 kDa) due to proteolysis within the B domain. The 90-kDa HCh reflects the absence of the B domain, whereas the 210-kDa species includes the intact B domain. The LCh (80 kDa) corresponds to the A3, C1, and C2 domains. The two chains are noncovalently linked by metal ions between the A1 and A3 domains (6, 7).FVIII is transformed into an active form by limited proteolysis by two essential serine proteases, thrombin and FXa (8, 9). This procoagulant activity is more pronounced after thrombin activation than after FXa activation (10). Cleavage at Arg 740 removes the size-heterogeneous B domain from the HCh, producing a 90-kDa HCh fragment, and further cleavage of the 90-kDa HCh fragment (A1-A2) at Arg 372 between the A1 and A2 domains produces 54-(A1) and 44-kDa (A2) species. Cleavage of the 80-kDa LCh fragment (A3-C1-C2) at Arg 1689 between the B and A3 domains removes 40 amino-terminal acidic peptides from the A3 domain and produces a 72-kDa fragment. A unique cleavage by FXa at Arg 1721 produces a 67-kDa LCh fragment. The active form of FVIII is a metal-linked heterotrimer consisting of the A1/A2/A3-C1-C2 domains, lacking the middle B domain (6). Cleavage at Arg 740 between the A2 and B domains does not contribute to the generation of the FVIII activity. In contrast, cleavages at Arg 372 and Arg 1689 are essential for optimal FVIII activity (11-13). Activation of human FVIII at its physiological concentration and at pH 7.4 is followed by a reduction in activity (14). This loss of activity is not caused by further proteolysis but by dissociation of the A2 domain from the active form of FVIII heterotrime...

“…Binding in this domain appears to be competitive and mutually exclusive (4,5,8,9). The C2 domain has also been shown to participate in binding to factor Xa and thrombin (10,11). Additionally, a major epitope for allo-and autoantibodies and for monoclonal antibodies has been located within the C2 domain (4,6,12), indicating that this region could be an antigenic "hot spot."…”

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confidence: 99%

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

Soeda

Nogami

Nishiya

et al. 2009

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Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dosedependently (K d ‫؍‬ 108 nM). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ϳ95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 proteasecleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228 -2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228 -2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.