Cleavage of Factor VIII Heavy Chain Is Required for the Functional Interaction of A2 Subunit with Factor IXa

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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: Methodsmentioning

confidence: 99%

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

confidence: 99%

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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“…Cleavage at Arg 372 exposes a cryptic functional factor IXainteractive site in the A2 domain (11), while cleavage at Arg 1689 liberates factor VIII from von Willebrand factor (12) and contributes to factor VIIIa specific activity (13,14), thus making both sites essential for procofactor activation. Although it is known that cleavage at Arg 740 represents a fast step relative to cleavage at other sites in the activation of factor VIII (15), the role for this event is unknown.…”

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confidence: 99%

“…Activation of factor VIII occurs through proteolysis by either protease via cleavage of three P1 residues at Arg 740 (A2-B domain junction), Arg 372 (A1-A2 domain junction), and Arg 1689 (a3-A3 junction) (6). After factor VIII activation, there is a weak electrostatic interaction between the A1 and A2 domains of factor VIIIa (9,10) and spontaneous inactivation of the cofactor occurs through A2 subunit dissociation from the A1/A3-C1-C2 dimer (11).…”

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confidence: 99%

Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII

Newell

Fay

2007

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Factor VIII is a plasma protein in which defects or deficiencies cause hemophilia A, the most frequently occurring severe inherited bleeding disorder. The activated form of factor VIII, factor VIIIa, functions as a cofactor for factor IXa, increasing its catalytic efficiency by several orders of magnitude in the phospholipid-and Ca 2ϩ -dependent conversion of factor X to factor Xa (1). Factor VIII is synthesized as an ϳ300-kDa single chain polypeptide (corresponding to 2332 amino acids) and is designated as six domains based on internal homologies: NH 2 -A1-A2-B-A3-C1-C2-COOH (2, 3). Moreover, preceding the A3 domain and following the A1 and A2 domains are short segments containing high concentrations of acidic residues designated a1 (residues 337-372), a2 (residues 711-740), and a3 (1649 -1689). Factor VIII is processed by cleavage at the B-A3 junction to generate a divalent metal ion-dependent heterodimeric protein composed of a heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains) (2-5).The inactive factor VIII procofactor is converted to factor VIIIa through limited proteolysis catalyzed by thrombin or factor Xa (6, 7). Thrombin is believed to act as the physiological activator of factor VIII, as association of factor VIII with von Willebrand factor impairs the capacity for the membrane-dependent factor Xa to efficiently activate the procofactor (6 -8). Activation of factor VIII occurs through proteolysis by either protease via cleavage of three P1 residues at Arg 740 (A2-B domain junction), Arg 372 (A1-A2 domain junction), and Arg 1689 (a3-A3 junction) (6). After factor VIII activation, there is a weak electrostatic interaction between the A1 and A2 domains of factor VIIIa (9, 10) and spontaneous inactivation of the cofactor occurs through A2 subunit dissociation from the A1/A3-C1-C2 dimer (11).Cleavage at Arg 372 exposes a cryptic functional factor IXainteractive site in the A2 domain (11), while cleavage at Arg 1689 liberates factor VIII from von Willebrand factor (12) and contributes to factor VIIIa specific activity (13, 14), thus making both sites essential for procofactor activation. Although it is known that cleavage at Arg 740 represents a fast step relative to cleavage at other sites in the activation of factor VIII (15), the role for this event is unknown. In the present study, cleavage at Arg 740 is examined using recombinant factor VIII variants possessing single point mutations of R740Q and R740H. Our results indicating altered rates of generation of factor VIIIa subunits dependent upon the residue at position 740 suggest an ordered mechanism for thrombin activation of factor VIII, whereby initial cleavage at EXPERIMENTAL PROCEDURESReagents-The monoclonal antibody 1A4 recognizing the A3 domain was a generous gift from Bayer. The anti-A2 domain factor VIII monoclonal antibody R8B12 (9) was obtained from Green Mountain Antibodies. The monoclonal antibody ESH8 (16) recognizing the C2 domain was purchased from American Diagnostica. The reagents human ␣-thrombin, factor IXa, f...

“…Cleavage at Arg-372 is a critical step in thrombin activation of factor VIII as it exposes a cryptic functional factor IXa-interactive site in the A2 domain (15), whereas cleavage at Arg-1689 liberates factor VIII from von Willebrand factor (16) and contributes to factor VIIIa specific activity (17,18). Although cleavage at Arg-740 represents a fast step relative to cleavages at other P1 residues in the activation of factor VIII (19), the influence of Arg-1689 cleavage on cleavages in the heavy chain remains unknown.…”

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Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

Newell

Fay

2009

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The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were ϳ50 and ϳ10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an ϳ340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed ϳ4-and 11-fold reductions in A2 subunit generation and ϳ3-and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.Factor VIII, a plasma protein missing or defective in individuals with hemophilia A, is synthesized as an ϳ300-kDa single chain polypeptide corresponding to 2332 amino acids. Within the protein are six domains based on internal homologies and ordered as NH 2 -A1-A2-B-A3-C1-C2-COOH (1, 2). Bordering the A domains are short segments containing high concentrations of acidic residues that follow the A1 and A2 domains and precede the A3 domain and are designated a1 (residues 337-372), a2 (residues 711-740), and a3 (1649 -1689). Factor VIII is processed by cleavage at the B-A3 junction to generate a divalent metal ion-dependent heterodimeric protein composed of a heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains) (3).The activated form of factor VIII, factor VIIIa, functions as a cofactor for factor IXa, increasing its catalytic efficiency by several orders of magnitude in the phospholipid-and Ca 2ϩ -dependent conversion of factor X to factor Xa (4). The factor VIII procofactor is converted to factor VIIIa through limited proteolysis catalyzed by thrombin or factor Xa (5, 6). Thrombin is believed to act as the physiological activator of factor VIII, as association of factor VIII with von Willebrand factor impairs the capacity for the membrane-dependent factor Xa to efficiently activate the procofactor (5, 7). Activation of factor VIII occu...