The Mutation Leu335Pro in Factor VIII Results in Its Rapid Inactivation by Thrombin-Catalyzed Proteolysis at Arg336.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2690-2690
Author(s):  
Fatbardha Varfaj ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Time Course ◽  
Specific Activity ◽  
Residual Activity ◽  
Factor Xa ◽  
Activity Data ◽  
Activity Increase ◽  
Thrombin Cleavage ◽  
Factor Viiia

Abstract Activation of the heterodimeric factor VIII procofactor is achieved by proteolysis of the heavy chain at residues Arg740 and Arg372 and of the light chain at Arg1689 in reactions catalyzed by thrombin or factor Xa. The active cofactor, factor VIIIa, is a heterotrimer consisting of the A1, A2, and A3-C1-C2 subunits. Factor VIIIa inactivation occurs by two mechanisms, spontaneous dissociation of the A2 subunit from factor VIIIa and proteolytic inactivation catalyzed by activated protein C (APC) as well as factor Xa. APC-catalyzed inactivation of factor VIIIa results from proteolysis at the P1 residues Arg336 and Arg562 within the A1 and A2 subunits, respectively, with cleavage at Arg336 representing the dominant reaction. Factor Xa-catalyzed inactivation of factor VIIIa occurs following cleavage only at the former site. While investigating the role for residues in the P2 position (residue 335) in the proteolytic mechanism for factor VIIIa inactivation, we stably expressed and purified several recombinant B-domainless factor VIII mutants. One variant, Leu335Pro, was based upon the optimal residue (Pro) at the P2 position for thrombin. Specific activity of the Leu335Pro factor VIII was ∼75% that of WT factor VIII. A time course of thrombin-catalyzed activation of the Leu335Pro factor VIII, as monitored by a one-stage clotting assay, yielded a similar activity increase to that of WT, consistent with similar rates of cleavages converting the procofactor to cofactor. However, this activity was highly unstable in the variant and quickly decayed such that at 10 min following thrombin addition, the residual activity of the variant was reduced ∼40-fold compared to WT. To investigate the reason for this rapid rate of inactivation of the newly generated cofactor, we monitored cleavages in the factor VIII heavy chain following reaction with thrombin, as well as APC and factor Xa by Western blot analysis. As predicted from the activity data, thrombin efficiently cleaved both the WT and Leu335Pro at Arg740 and Arg372, generating the A1 and A2 subunits of factor VIIIa. In WT factor VIIIa, we also observed a relatively slow rate of cleavage in the A1 subunit generating a product consistent with proteolysis at the APC-sensitive Arg336 site. This material represented <20% of the total A1 subunit at an extended time point (40 min) in the reaction suggesting that thrombin-cleavage at this site is a minor pathway. However, thrombin cleavage of the A1 subunit derived from Leu335Pro generated the truncated A1 product at a >50-fold increased rate compared with WT. Since Pro represents an optimal residue at the P2 position for thrombin action, these results suggest that the presence of this residue facilitated cleavage at Arg336. While the presence of Pro335 did not appreciably affect cleavage of Arg336 by APC compared to the cleavage rate observed in the WT factor VIII, this rate was ∼5-fold less than thrombin-catalyzed cleavage at Arg336 in the Leu335Pro variant. Furthermore, the rate of factor Xa-catalyzed cleavage at this site in the variant was accelerated ∼10-fold relative to WT. Overall, these data suggest that replacement of Leu335 with Pro markedly increases proteolytic inactivation of factor VIIIa by both thrombin, and to a lesser extent factor Xa by creating a more optimal region for active site engagement at Arg336.

Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1692-1692
Author(s):  
Jennifer Newell ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Time Course ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1711-1711 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Timothy Myles ◽  
Lawrence L. Leung ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Active Site ◽  
Specific Activity ◽  
Solid Phase ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Marginal Effect ◽  
Thrombin Cleavage ◽  
A2 Domain

Abstract Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa In Vivo

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 176-183 ◽  
Author(s):  
S.W. Pipe ◽  
A.N. Eickhorst ◽  
S.H. McKinley ◽  
E.L. Saenko ◽  
R.J. Kaufman
Keyword(s):  
Factor Viii ◽  
Missense Mutation ◽  
Time Course ◽  
Hemophilia A ◽  
Specific Activity ◽  
Metabolic Labeling ◽  
Two Stage ◽  
One Stage ◽  
Thrombin Cleavage

Abstract Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation whereARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing theARG531HIS mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531HIS was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531HISdemonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,ARG531HIS had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII WT). Partially purified FVIII WT and ARG531HISproteins were subjected to functional activation by incubation with thrombin. ARG531HIS demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531HIS missense mutation predicts a charge change to the A2 subunit, we hypothesized that theARG531HIS A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster forARG531HIS compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.

Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1725-1725 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Activity Increase ◽  
Wide Range

Abstract We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

scholarly journals Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1664-1672 ◽  
Author(s):  
DP O'Brien ◽  
JK Pattinson ◽  
EG Tuddenham
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Time Course ◽  
Hemophilia A ◽  
Severe Hemophilia ◽  
Thrombin Cleavage ◽  
Before And After ◽  
Functional Defect ◽  
Thrombin Activation

We have purified factor VIII from a patient with moderately severe hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein to Western blot analysis after time course activation with thrombin. The cross reacting material-positive (CRM+) FVIII has the normal distribution of heavy and light chains before thrombin activation, and, after incubation with the enzyme, appropriate cleavages are made at positions 740 and 1689. However, the normal thrombin cleavage at position 372 in the heavy chain of this molecule does not occur. This result is consistent with the demonstration in the patient's leukocyte DNA of a C to T transition in codon 372, leading to the substitution of a cysteine for an arginine residue at the heavy chain internal cleavage site. The severely impaired functional activity of this molecule confirms that the heavy chain of FVIII must be proteolysed in order to effect full cofactor activation in vivo. However, a threefold activation was detected when this protein was incubated with thrombin. No evidence of thrombin-mediated cleavage at position 336 in the heavy chain was detected, in contrast to the variant recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation studies of the FVIII/von Willebrand factor (vWF) complex before and after thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild type FVIII dissociates from vWF after cleavage by the enzyme. In contrast, incomplete dissociation was detected in the case of FVIII 372-Cys. We conclude that the functional defect in FVIII 372-Cys is a consequence of the resistance to proteolysis of the internal scissile bond in the heavy chain.

Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 847-847
Author(s):  
Jennifer L. Newell ◽  
Amy E. Griffiths ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Cleavage Site ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Factor Xa ◽  
Wild Type ◽  
Cleavage Rate ◽  
Thrombin Cleavage ◽  
Type Factor ◽  
Bold Type

Abstract Abstract 847 Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are 370QIR*↓SVA375, 1687SPR*↓SFQ1692, and 738EPR*↓SFS743, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor. Disclosures: No relevant conflicts of interest to declare.

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

2001 ◽  
Vol 276 (15) ◽  
pp. 12434-12439 ◽  
Author(s):  
Philip J. Fay ◽  
Maria Mastri ◽  
Mary E. Koszelak ◽  
Hironao Wakabayashi
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Fluorescence Anisotropy ◽  
Factor Xa ◽  
Factor X ◽  
Functional Interaction ◽  
Factor Ixa ◽  
Factor Viiia ◽  
A2 Domain ◽  
Stimulation Of

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

Enhancement of Factor VIII Stability by Replacing Multiple Charged Residues at the A2 Domain Interface

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1016-1016
Author(s):  
Hironao Wakabayashi ◽  
Amy E Griffiths ◽  
Philip Fay
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Specific Activity ◽  
Factor Xa ◽  
Fold Increase ◽  
Decay Rates ◽  
Single Mutation ◽  
Factor Viiia ◽  
Group A ◽  
Group B

Abstract Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665, or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 2008, in press). These mutations did not appreciably affect factor VIII specific activity or thrombin generation parameters. Factor VIII structure studies suggest D519 is buried at the A1/A2 domain interface, while E665 and E1984 localize at the A2/A3 domain interface. In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effect of these mutations. Factor VIII variants generated included double mutants with a mutation at D519 and mutation at either E665 or E1984 (Group A), double mutants with a mutation at E665 and mutation at E1984 (Group B), and triple mutants with a mutation at D519 and mutations at both E665 and E1984 (Group C). Most of the mutants retained normal specific activity values compared to wild type (WT) with exceptions noted for E665A/E1984A, E665A/E1984V and D519V/E665V/E1984V which showed ~2-fold reductions in this parameter. Studies assessing factor VIII stability involved monitoring the rates of loss of factor VIII activity by factor Xa generation assay following incubation of factor VIII (4 nM) at 55ºC. The rate of decay of factor VIIIa was monitored over time at 23ºC using the factor Xa generation assay following activation of factor VIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. The Group A variants D519A/E665A, D519A/D665V, and D519V/E665V showed significant enhancement (up to ~1.3-fold for the D519A/D665V variant) in factor VIII thermal stability as compared with the best single mutation in that pairing, and representing actual decay rates that approached 45% the WT value. On the other hand, the relative factor VIII decay rates for three of the four of the Group B mutants were somewhat increased compared with the best single mutation in the pairing. No significant changes were observed for the Group C mutants. Evaluation of factor VIIIa stability revealed large enhancements of up to ~4-fold compared with the single mutants for all of the Group A variants. Group B variants yielded poorer results when compared with the better individual mutation in the pairing. The triple mutations (Group C) showed the largest factor VIIIa stability enhancements with maximal stability observed for D519V/E665V/E1984A, which showed a decay rate that was ~10% the WT value. A calibrated thrombin generation assay using a fluorogenic substrate was performed on selected mutants using a sub-physiologic factor VIII concentration (0.2 nM). Enhancements in selected parameter values were observed for the D519V/E665V variant (~2.3-fold increase in the peak height and ~1.5-fold increase in endogenous thrombin potential compared with WT), while the D519A/E665V, D519V/E1984A, and D519V/E665V/E1984A variants showed 1.2 to 1.7-fold increases in these parameter values. These observations may suggest a greater capacity for thrombin generation per unit concentration factor VIII for these variants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield factor VIII reagents with improved stability parameters.

FVIII Heavy Chain Enhances Tenase Activity Induced By FVIIIa Mimicking Bispesific Antibody, ACE910

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1481-1481
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Takehisa Kitazawa ◽  
Kunihiro Hattori ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Research Funding ◽  
Factor Xa ◽  
Factor X ◽  
Advisory Committees ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Physiological Structure

Abstract Background: ACE910, asymmetric bispecific monoclonal antibodies to activated factor IX (IXa) and factor X, mimics the cofactor function of activated factor VIII (VIIIa) by modulating an optimal position on the tenase assembly. The estimated therapeutic range of ACE910 shows ~30% of thrombin generation in native tenase assembly, supporting that the structure on ACE910-mimicking tenase assembly is different from that on native tenase. Being close to physiological structure consisting from factor IXa, factor X, and factor VIIIa is important for potentiating the clotting function. We examined the effects of factor VIII subunits (light chain, heavy chain, A1 and A2, C2) on ACE910-tenase. Materials/Methods: The factor VIII light chain and heavy chain were isolated from EDTA-treated recombinant factor VIII following chromatography on SP- and Q- Sepharose columns. The A2 and A1 subunits were purified from thrombin-cleaved factor VIII heavy chain by Heparin-, SP- Sepharose columns. Purified factor Xa generation assays was examined with (i) factor VIII subunit (0-40 nM), ACE910 (10 µg/ml), phospholipid (PL) (40 µM), factor IXa (1 nM) and factor X (200 nM), (ii, iii) the A2 or heavy chain (40 nM), ACE910 (10 µg/ml), PL (40 µM), factor IXa and factor X (1 or 0-80 nM, and 0-300 or 200 nM, respectively). These mixtures were reacted for five minutes (i, ii) or one minute (iii). These assays were conducted at 37 °C. Results: (i) The factor Xa generation in ACE910-tenase complex in the absence of factor VIIIa was 10.1±2.2 nM. With the intact heavy chain and A2, amounts of factor Xa were increased dose-dependently, resulting in 1.3- and 1.2-fold increases, respectively. While, the light chain and A1 subunit failed to increase at all. (ii) Vmax for factor X in ACE910-tenase was 173.0±7.0 nM and Km was 31.2±3.9 nM. Vmax obtained with the heavy chain or A2 was 175.9±6.1 or 159.0±6.1 nM, whilst Km was 17.0±2.2 or 31.9±3.5 nM, respectively, indicating that the heavy chain enhanced the binding affinity for factor X in ACE910-tenase. (iii) Vmax for factor IXa in ACE910-tenase was 43.8±2.7 nM and Km was 36.9±4.8 nM. With the heavy chain or A2, Vmax was 46.8±3.0 or 45.0±3.1 nM, and Km was 36.4±3.0 or 32.1±4.9 nM, respectively, indicating that either the heavy chain or A2 did not enhance the catalytic activity and the binding affinity for factor IXa in ACE910-tenase. Conclusion: ACE910-tenase assembly seems to be close to physiological structure by the presence of intact heavy chain interacting with factor X. In addition, ACE910 may substitute the position such as the factor VIII(a) light chain associated with FIXa and FX on ACE910-tenase assembly defecting factor VIII. Disclosures Minami: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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