Identification of a Protein S-Interactive Site on the Factor VIII A2 Domain.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2684-2684
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Tetsuhiro Soeda ◽  
Akira Yoshioka ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Activated Protein C ◽  
Protein S ◽  
Cross Linking ◽  
Factor V ◽  
Dependent Manner ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Mutant Forms ◽  
A2 Domain

Abstract Protein S functions as a cofactor of activated protein C that inactivates factor VIII(a) and factor V(a). We recently have reported a new regulatory mechanism that protein S interacted with both the A2 and A3 domains in factor VIII, and consequently this cofactor directly impaired the factor Xase complex by competing the interaction of factor IXa to factor VIIIa (Blood2006; 108, 487a). Since factor IXa blocked the binding of A2 subunit to protein S, we attempted several approaches to localize the protein S-interactive site(s) on the factor VIII A2 domain. An anti-A2 monoclonal antibody (mAb413) with the 484–509 epitope, recognizing a factor IXa-interactive site on the A2, inhibited the A2 binding to immobilized protein S up to approximately 90% in a dose-dependent manner in a surface plasmon resonance-based assay. Furthermore, ELISA-based assay showed that a synthetic peptide corresponding to residues 484–509 directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. The cross-linked product formed with EDC was consistent with 1:1 stoichiometry of reactants, suggesting specificity in the 484–509 peptide and protein S interaction. This cross-linking formation was blocked by the addition of the 484–497 peptide, whilst not by the 498–509 peptide, supporting the presence of protein S-interactive site within residues 484–497. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S product showed that three sequential basic residues (S488, R489 and R490) could not be detected, supporting that three residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for protein S-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in baculovirus system and purified. Compared with wild type A2 (Kd: ∼9 nM), each binding affinity of S488A, R489A, or R490A A2 mutant for protein S was decreased by 4∼5-fold (32, 40 and 40 nM, respectively). These results indicate that the 484–509 region in the factor VIII A2 domain, and in particular a cluster of basic amino acids at residues 488–490, contributes to a unique protein S-interactive site.

Identification of a protein S-interactive site within the A2 domain of the factor VIII heavy chain

2009 ◽  
Vol 102 (10) ◽  
pp. 645-655 ◽  
Author(s):  
Masahiro Takeyama ◽  
Evgueni L. Saenko ◽  
Katsumi Nishiya ◽  
Kenichi Ogiwara ◽  
Midori Shima ◽  
...  
Keyword(s):  
Solid Phase ◽  
Activated Protein C ◽  
Protein S ◽  
Cross Linking ◽  
Binding Assays ◽  
Functional Region ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Solid Phase Binding ◽  
A2 Domain

SummaryWe have recently demonstrated that protein S impairs the intrinsic tenase complex, independent of activated protein C, in competitive interactions between the A2 and A3 domains of factor VIIIa and factor IXa. In the present study, we have identified a protein S-interactive site in the A2 domain of factor VIIIa. Anti-A2 monoclonal antibody recognising a factor IXa-functional region (residues 484–509) on A2, and synthetic peptide inhibited the A2 binding to protein S by ∼60% and ∼70%, respectively, in solid-phase binding assays. The 484–509 peptide directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). The cross-linked adduct was consistent with 1:1 stoichiometry of reactants. Cross-linking formation was blocked by addition of the 484–497 peptide, but not by the 498–509 peptide. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S adduct showed that three sequential residues (S488, R489, and R490) in A2 were not identified, suggesting that these residues participate in cross-link formation. Mutant A2 molecules where these residues were converted to alanine were evaluated for the binding of protein S. The S488A, R489A, and R490A mutants demonstrated ∼four-fold lower affinity than wild-type A2.These results indicate that the 484–509 region in the A2 domain of factor VIIIa, in particular sequential residues at positions 488–490, contributes to a unique protein S-interactive site.

Synergistic Cofactor Function of Factor V and Protein S to Activated Protein C in the Inactivation of the Factor Villa – Factor IXa Complex

1997 ◽  
Vol 78 (03) ◽  
pp. 1030-1036 ◽  
Author(s):  
Lei Shea ◽  
Xuhua He ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Species Specificity ◽  
Factor V ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Cofactor Activity ◽  
Apc Protein ◽  
Bovine Factor

SummaryHuman factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine.origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.

Contribution of P4-P3′ Residues Surrounding P1 Residues Arg336 and Arg562 in the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1693-1693
Author(s):  
Fatbardha Varfaj ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Catalytic Mechanism ◽  
Specific Activity ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Minor Role ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Fold Reduction

Abstract The essential role of factor VIII in blood coagulation is evident from the bleeding diathesis hemophilia A, which results from a deficiency or defect in factor VIII. Activated factor VIII (factor VIIIa) serves as a cofactor for factor IXa in the factor Xase complex, which activates factor X during the propagation phase of coagulation. Factor VIIIa is a non-covalent heterotrimer consisting of A1, A2 and A3–C1–C2 subunits. Down-regulation of factor Xase is achieved by cofactor inactivation and is thought to occur by a non-proteolytic mechanism involving dissociation of the A2 subunit as well as a proteolytic mechanism catalyzed by activated protein C (APC). APC cleaves the P1 residues Arg336 near the C-terminus of the A1 subunit and Arg562 bisecting the A2 subunit. We recently demonstrated that these cleavages occur in an independent non-sequential fashion, with residue Arg336 being cleaved at a rate ~25-fold faster than Arg562 (Varfaj et al., Biochem J. 2006). While substantial evidence implicates involvement of exosite-directed interactions in the catalytic mechanism of APC, another factor that may contribute to the disparate cleavage rates are residues surrounding the P1 Arg residues. To examine the roles of these sequences in cofactor cleavage, we prepared two factor VIII mutants where the P2–P4 and P1′–P3′ residues surrounding Arg336 (Pro-Gln-Leu and Met-Lys-Asn, respectively) were replaced with those residues surrounding Arg562 (Val-Asp-Gln and Gly-Asn-Gln, respectively), and designated Arg336P2-P4A2 and Arg336P1′-P3′A2. In addition, a single mutant was prepared where the P4-P3′ residues surrounding Arg562 were replaced with those residues surrounding Arg336, and designated Arg562P4-P3′A1. Recombinant, B-domainless factor VIII proteins were stably expressed in BHK cells and purified. Specific activity values measured for Arg336P2-P4A2 and Arg336P1′-P3′A2 mutants were similar to that of wild type (WT) factor VIII, whereas Arg562P4-P3′A1 showed a specific activity value <1% that of WT factor VIII. This latter observation was consistent with the substitution of the A1 residues altering the factor IXa-interactive site contained within A2 residues 558–565. Western blot analysis examining the rates of APC-catalyzed cleavage at Arg336 showed an ~60-fold reduction for the Arg336P2-P4A2 mutant and an ~10-fold reduction for Arg336P1′-P3′A2 mutant compared to cleavage rates observed for WT factor VIIIa. Rates of cleavage at the Arg562 site in these mutants were similar to the WT protein at this site. These results suggest that the native sequence surrounding Arg336 possesses residues more optimal for cleavage by APC than those that surround Arg562 in the A2 subunit. Examination of the Arg562P4-P3′A1 mutant showed a modest increase (~2-fold) in cleavage rate at Arg562, whereas cleavage at the Arg336 was similar to the WT control. Overall, these results suggest a relatively minor role for specific sequences in the cleavage mechanism for APC at the A2 site, whereas a more dominant role for sequence specificity appears necessary for efficient proteolysis at the A1 site, which represents the primary site of attack by APC.

Factor V as Anticoagulant Cofactor for Activated Protein C in Factor Va Inactivation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3075-3075
Author(s):  
Thomas J Cramer ◽  
John H. Griffin ◽  
Andrew J. Gale
Keyword(s):  
Protein C ◽  
Time Course ◽  
Activated Protein C ◽  
Protein S ◽  
Anticoagulant Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Anticoagulant Function ◽  
Apc Protein

Abstract Factor V (FV) is a cofactor that promotes inactivation of activated factor VIII (FVIIIa) by the activated protein C and protein S complex (APC/protein S). Cleavage in FV at Arg506 is required for proteolytic inactivation of FVa, but also for the anticoagulant function of FV as cofactor for APC in the inactivation of FVIIIa. This is demonstrated by the well known FVLeiden mutant with Arg506 mutated to glutamine (Q506), causing APC resistance due to both impaired sensitivity of Q506FVa to APC and reduced cofactor activity of Q506FV for APC inactivation of FVIIIa. However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. Thus, we question whether the APC-mediated inactivation of FVIIIa is relevant to the FVLeiden thrombophilic phenotype. Rather, we hypothesized that FV can function as an anticoagulant cofactor for the APC/protein S complex in the inactivation of activated FV (FVa). To test this hypothesis, we designed a coagulation assay initiated by tissue factor that was sensitive to FV but was insensitive to FVIII. FV was titrated into FV deficient plasma and clotting times were measured in absence and presence of APC to determine an APC sensitivity ratio (APCsr). An increase in the APCsr was observed as the level of FV was increased, suggesting an anticoagulant function of FV. Similar titrations were done with Q506FV, showing no increase in clotting time when APC was present and an APCsr of 1.0 in the presence of Q506 FV. Control experiments confirmed that this clotting assay was insensitive to the presence or absence of FVIII; thus, these assays were reflecting FVa inactivation. The potential anticoagulant effect of FV as cofactor for APC in FVa inactivation was further investigated by monitoring proteolysis of purified FVa by APC over time using SDS PAGE. Recombinant purified FVa was labeled with a fluorescent dye, and then subjected to proteolysis by APC/protein S in the absence or presence of FV in a time course. The resulting FVa fragments seen on SDS gels reflected the known cleavages at Arg306 and Arg506, and the FVa cleavage products were quantified by digital fluorescent scanning of the gel. FV stimulated a small but statistically insignificant increase in the rate of FVa cleavage by APC/protein S. Thus, in our experimental conditions, we found a significant anticoagulant effect of FV in clotting assays that were sensitive to FV but not sensitive to FVIII whereas in purified reaction mixtures there was not a significant enhancement by FV of APC proteolysis of FVa. These data contrasting FV’s apparent APC-cofactor activities between plasma and purified reaction mixtures lead us to speculate that other factors or mechanisms present in plasma also contribute to the anticoagulant function of APC in a FV dependent manner.

Partial Reconstitution of Factor VIII Activity from a Mild Crm+ Hemophilia A Patient by Replacement of the Defective A2 Domain

1998 ◽  
Vol 79 (05) ◽  
pp. 943-948 ◽  
Author(s):  
W. C. Pieneman ◽  
P. Fay ◽  
E. Briët ◽  
P. H. Reitsma ◽  
R. M. Bertina
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Wild Type ◽  
Coding Region ◽  
Factor Viii Activity ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor Viii Antigen ◽  
A2 Domain ◽  
Reduced Activity

SummaryWe further characterised the abnormal factor VIII molecule (factor VIII Leiden) of a Crm+, mild hemophilia A patient with a factor VIII activity of 0.18 IU/ml and a factor VIII antigen of 0.95 IU/ml. Mutation analysis of the coding region, promoter and 3’ untranslated region of the factor VIII gene revealed the presence of a C to T substitution at codon 527. This nucleotide change predicts the replacement of an arginine to tryptophan in the A2 domain close to a suggested binding site for factor IXa. Since a previous study of this mutant factor VIII protein suggested that this protein had a reduced affinity for factor IXa, position 527 in the protein might be involved in the interaction with factor IXa.In this study we gathered evidence for our hypothesis that the Arg to Trp mutation at position 527 is the cause of the reduced activity of factor VIII Leiden. Replacement of the mutated A2 domain by wild type A2 domain partially corrected the defect.Factor VIII from normal and factor VIII Leiden plasma was concentrated by cryoprecipitation, activated with thrombin and incubated with excess wild type A2 domain. Competition with excess isolated human A2 domain resulted in a partial reconstitution of the factor VIIIa activity of thrombin treated factor VIII Leiden. This supports the hypothesis that the mutation in the A2 domain is the cause of the reduced factor VIII activity.

Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Dependent Manner ◽  
C2 Domain ◽  
Factor X ◽  
Calcium Dependent ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Gla Domain

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

C4b-binding Protein Inhibits the Factor V-dependent but not the Factor V-independent Cofactor Activity of Protein S in the Activated Protein C-mediated Inactivation of Factor VIIIa

2001 ◽  
Vol 85 (05) ◽  
pp. 761-765 ◽  
Author(s):  
Robbert van de Poel ◽  
Joost Meijers ◽  
Bonno Bouma
Keyword(s):  
Protein C ◽  
Binding Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Coagulation Factors ◽  
Factor V ◽  
Factor Viiia ◽  
S Factor ◽  
Cofactor Activity ◽  
Apc Protein

SummaryActivated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equi-molar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.

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.

Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1714-1714
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Kohei Tatsumi ◽  
Yuri Fujita ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Protein S ◽  
Factor X ◽  
Cleavage Rate ◽  
Physiological Concentration ◽  
C2 Domains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Km Value

Abstract Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.

scholarly journals Activated Protein C has a Regulatory Role in Factor VIII Function

Blood ◽  
2021 ◽  
Author(s):  
Amelia R. Wilhelm ◽  
Nicole A. Parsons ◽  
Benjamin J Samelson-Jones ◽  
Robert J Davidson ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Inhibitory Antibody ◽  
Apc Resistance ◽  
Fv Leiden ◽  
A2 Domain ◽  
Tail Clip

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII and biochemical studies suggest APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein to an APC resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated a dose-dependent sensitivity to APC with or without protein S, while FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately 5-fold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, the tail clip assay was performed in homozygous HA/FV-Leiden mice infused with FVIII-QQ or FVIII-WT in the presence or absence of mAb1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FV-Leiden mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, supporting the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.

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