Kinetics of Factor VIII Light‐Chain Cleavage by Thrombin and Factor Xa

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...

Section: Characterization Of Recombinant Factor VIII Protein-supporting

confidence: 80%

mentioning

confidence: 99%

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

Newell

Fay

2009

“…Cleavage at Arg 1689 in Factor VIII Proteins by ThrombinPrevious results have suggested that the presence of heavy chain promotes thrombin proteolysis of light chain (14). The above observations monitored using the anti-A2 domain antibody support this contention.…”

Section: Characterization Of Recombinantsupporting

confidence: 73%

“…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.…”

mentioning

confidence: 99%

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

Newell

Fay

2007

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...

“…A monoclonal antibody directed against this region inhibited thrombin activation of factor VIII (51,52), and recombinant B-domainless factor VIII molecules in which this region was partially deleted were observed to require higher thrombin concentrations for efficient activation of the procofactor (52,53). Furthermore, in experiments using a factor VIII chimera where A2 residues 712-736 were replaced with a high affinity thrombin site from the serpin, heparin cofactor II showed increased rates of both cleavages at Arg 372 by thrombin and overall factor VIII activation (54), suggesting that the C-terminal region of A2 influences cleavages at distal sites in the factor VIII molecule.…”

Section: Discussionmentioning

confidence: 99%

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Nogami

Zhou

Myles

et al. 2005

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser 205 3 Ala mutant indicated a high affinity site in the A1 subunit (K d ϳ5 nM) that was dependent upon the Na ؉ -bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K d ϳ100 nM) was observed for both Na ؉ -bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719 -740, blocked thrombin cleavage of the isolated heavy chain at Arg 372 and Arg 740 and inhibited A2 binding to thrombin Ser 205 3 Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp 392 3 Ala/Asp 394 3 Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg 740 , whereas cleavage at Arg 372 was not affected. These data suggest the acidic region comprising residues 389 -394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg 740 during procofactor activation.