Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking

1998 ◽  
Vol 80 (09) ◽  
pp. 418-422 ◽  
Author(s):  
Kirsty Lapan ◽  
Philip Fay
Keyword(s):  
Serine Protease ◽  
Heavy Chain ◽  
Solid Phase ◽  
Activated Protein C ◽  
Cross Linking ◽  
Factor X ◽  
Serine Protease Domain ◽  
C Terminus ◽  
Factor Viiia ◽  
Solid Phase Binding

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

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.

Identification Of The Thrombin-Binding Site On Factor VIII Regulating Arg372 Cleavage In The Factor VIII Heavy Chain

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3570-3570
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Koji Yada ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Solid Phase ◽  
Factor Ix ◽  
Cross Linking ◽  
Dependent Manner ◽  
Factor X ◽  
Dose Dependent

Abstract Factor VIII is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin. Activated factor VIII (VIIIa) forms the tenase complex and markedly amplifies the activation of factor X as a cofactor of factor IX. We had demonstrated that thrombin interacts with factor VIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008). The A2 residues 484-509 partially contribute to cleavage at Arg372 by thrombin, however, the major thrombin binding-site(s) regulating cleavage at Arg372 is unclear. Thrombin recognizes macromolecular substrates and cofactors through either or both of two anion-binding exosite I and II (ABE-I and -II), which are characterized by a high density of solvent-exposed basic residues. ABE-I binds to fibrinogen and hirudin (residues 54-65), whilst ABE-II is primarily characterized as the heparin-binding exosite. The A1 domain of factor VIII also binds to thrombin through the ABE-I (Nogami K. JBC 2005). In this study, we attempted to identify the thrombin-binding region on A1, and focused on the A1 residues 340-350, involving the clustered acidic residues and similar sequences of hirudin (residues 54-65). A synthetic peptide corresponding to the A1 residues 340-350 with sulfated Tyr346 (340-350-S(+)) was prepared to investigate factor VIII interaction with thrombin. Activation of factor VIII (100 nM) by thrombin (0.4 nM) with various concentrations of peptide was evaluated by measurement of factor VIIIa activity in a one-stage clotting assay. A 340-350-S(+) peptide showed a dose-dependent inhibition (by ∼60%) of thrombin-catalyzed activation, and the IC50 was 75 µM. A non-sulfated peptide also showed a modest inhibition by ∼40% (IC50 >400 µM), however. An experiment using thrombin substrate S-2238 demonstrated that P340-350-S(+) did not affect the thrombin activity. The effect of 340-350-S(+) peptide on the thrombin-catalyzed cleavage of heavy chain was further examined by SDS-PAGE/western blotting.The peptide significantly blocked the cleavage at Arg372 in a timed- and dose-dependent manner (IC50; 150 µM), whilst of interest the cleavage at Arg740 was little affected. A non-sulfated peptide also delayed the cleavage at Arg372, with a modest fast cleavage compared to sulfated one. The peptide did not inhibit factor FXa-catalyzed reaction to factor VIII. Direct binding of 340-350-S(+) peptide to thrombin was examined by a surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based solid phase assay, thrombin bound to immobilized 340-350-S(+) peptide with high affinity (Kd; 1.13 nM). EDC cross-linking fluid phase assay similarly revealed that formation of EDC cross-linking product between the biotinylated 337-350-S(+) peptide and thrombin were observed, and this cross-linking was completely inhibited by non-labeled 340-350-S(+) peptide (IC50; 1.0 µM). Taken together, we demonstrated that the A1 residues 340-350 (NEEAED(sY)DDDL) involving sulfated Tyr346 contained the thrombin binding-site responsible for the proteolytic cleavage at Arg372 in factor VIII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interacting Regions in the A1 and A2 Subunits of Factor VIIIa Identified by Zero-Length Cross-Linking

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3943-3950 ◽  
Author(s):  
Lynn M. O'Brien ◽  
Christine F. Huggins ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Activated Protein C ◽  
Salt Bridge ◽  
Cross Linking ◽  
C Terminus ◽  
Factor Viiia ◽  
Cross Linkage ◽  
Covalently Linked ◽  
Necessary And Sufficient ◽  
Acidic Region

Abstract Factor VIIIa is a heterotrimer of A1, A2, and A3-C1-C2 subunits, the activity of which is labile due to a weak affinity interaction of the A2 subunit with the A1/A3-C1-C2 dimer. We have used the zero-length cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), to localize regions of interaction within the A1 and A2 subunits. Reaction of factor VIIIa with EDC resulted in the formation of a cross-linked product of approximately 90 kD consisting of the A1 and A2 subunits as judged by Western blotting. Alkaline resistance of this product indicated an amide rather than ester linkage. Factor VIIIa activity decreased as the concentration of cross-linked product increased, suggesting that flexibility in the inter-subunit interaction may be required for proper cofactor function. This product was not formed in the contiguous A1-A2 domains of factor VIII, suggesting that, upon cofactor activation, a conformational change occurs that leads to the formation of a new interdomainal salt bridge(s). Reaction of the EDC-treated factor VIIIa with activated protein C (APC), which cleaves the A1 subunit at Arg336 and bisects the A2 subunit at Arg562, resulted in the formation of an approximately 30 kD product that contains the C-terminus region of A1 covalently linked to the N-terminal half of the A2. The approximately 90 kD cross-linked product was generated after reaction of A2 subunit with A1/A3-C1-C2 dimer but not with A1336/A3-C1-C2, a form of the dimer produced by APC cleavage and lacking the C-terminal acidic region of A1. A synthetic peptide corresponding to this acidic region (Met337-Arg372) was found to covalently cross-link to the isolated A2 subunit in 1:1 stoichiometry, suggesting that this region is both necessary and sufficient for the interaction of the A1 and A2 subunits. Sequence analysis of this product suggested that Glu344 in the A1 peptide may contribute to the cross-linkage. These results indicate that activation of factor VIII results in formation of a new ionic linkage(s) localized to the acidic C-terminal region of A1 and the N-terminal half of A2.

scholarly journals Active site-specific immunoassays

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 755-766 ◽  
Author(s):  
KG Mann ◽  
EB Williams ◽  
S Krishnaswamy ◽  
W Church ◽  
A Giles ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Solid Phase ◽  
Activated Protein C ◽  
Factor Xa ◽  
Immune Recognition ◽  
Factor X ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Active Site Histidine

Abstract This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

scholarly journals Active site-specific immunoassays

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 755-766 ◽  
Author(s):  
KG Mann ◽  
EB Williams ◽  
S Krishnaswamy ◽  
W Church ◽  
A Giles ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Solid Phase ◽  
Activated Protein C ◽  
Factor Xa ◽  
Immune Recognition ◽  
Factor X ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Active Site Histidine

This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

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.

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.

Mapping Factor VIIIa Inter-Subunit Interactions Using Chemical Modification and Zero-Length Cross-Linking.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1016-1016
Author(s):  
Stephen M. Miles ◽  
Jan Freas ◽  
Philip J. Fay
Keyword(s):  
Chemical Modification ◽  
Protein Modification ◽  
Cross Linking ◽  
Factor X ◽  
Cross Link ◽  
Subunit Interactions ◽  
Predictive Tool ◽  
Proteolytic Activation ◽  
Peptide Modification ◽  
Factor Viiia

Abstract Activated factor VIII (FVIIIa) is a complex of three subunits, designated A1, A2, and A3C1C2, that serves as a cofactor for factor IXa in the proteolytic activation of factor X. The structure and surface interactions between factor VIIIa subunits have yet to be fully defined, but a homology-based model is available as a predictive tool (Stoylova-McPhie et al, Blood, 2002). To investigate FVIIIa inter-subunit interactions we used a chemical modification approach to determine surface-exposed and potentially interactive residues. The protein modification reagents acetic anhydride and diethylpyrocarbonate (DEPC), that modify lysine and histidine residues, respectively, were used to identify residues that are modified in the free subunits but protected in the bound complex. The modified samples were digested with various proteases and the resulting digests were analyzed by MALDI-TOF mass spectrometry to determine sites of peptide modification (Figure 1). The majority of data observed was in agreement with the A1/A3C1C2 interface in the predicted model. Protection was observed at Lys-89, Lys-142, and Lys-230 in the A1 subunit, all of which are near the A3C1C2 interface. While most His residues were completely modified by DEPC, partial protection was seen at His-1954, His-1957, and His-1961 in A3C1C2, which we have previously shown to be an A1-interactive region (Ansong and Fay, Biochemistry, 2005). In addition to this data, we attempted to define inter-subunit contacts by covalently cross-linking the FVIIIa complex. Using the zero-length cross-linker 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), which cross-links Glu/Asp residues to Lys, four distinct cross-linked bands were identified by SDS-PAGE (Figure 2). The composition of the cross-linked products was determined using proteolytic digestion and MALDI analysis. The four products correspond to each possible combination of the three subunits. Interestingly, there are no candidate cross-link sites between A1 and A2 in the model, yet this cross-link is the most predominant of the four. One alternative is that the A1–A2 cross-link may involve the A1 337–372 region that is not represented in the model. Taken together, the data are directed toward physically defining interactive regions between FVIIIa subunits and serve to test and supplement current structure models. FIGURE 1, FIGURE 2 FIGURE 1, FIGURE 2.

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