Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dosedependently (K d ‫؍‬ 108 nM). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ϳ95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 proteasecleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228 -2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228 -2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

IntroductionFactor VIII (FVIII) is a glycoprotein cofactor that serves as a critical component in the intrinsic coagulation pathway. 1 Insufficient expression of FVIII or expression of nonfunctional FVIII results in hemophilia A, one of the most common severe hereditary bleeding disorders. Mature FVIII is synthesized as a single-chain polypeptide consisting of 2332 amino acid residues. 2,3 Based on internal sequence homologies, 3 types of structural domains have been identified, arranged in the order of A1-A2-B-A3-C1-C2. 4 FVIII circulates in plasma as a heterodimer of a heavy chain, consisting of the A1 (1-372) and A2 (373-740) domains, together with heterogeneous fragments of partially proteolyzed B domains , and a light chain (LCh) consisting of the A3 (1649-2019), C1 (2020-2172), and C2 (2173-2332) domains. 3,4 Thrombin and factor Xa (FXa) are the major physiologic enzymes that activate FVIII. Both enzymes cleave FVIII at Arg372, Arg740, and Arg1689 and transform it to its active form, an A1/A2/A3-C1-C2 heterotrimer. 5 Activated FVIII is incorporated into the FXase complex assembled on a phospholipid (PL) membrane surface and thus acts as an accelerator of FXa generation. Activated protein C (APC) inactivates activated FVIII by proteolytic cleavage at Arg336 in the presence of negatively charged PL. 5 In this reaction, the 54-kd A1 fragment is cleaved into a 40-kd fragment with a loss of the carboxy-terminal acidic region. The APC binding site in the FVIII molecule has been located within the residues 2009 to 2018 in the A3 domain of the LCh. 6 The site has not been confirmed in direct binding experiments, however.FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (VWF). 7 VWF regulates the synthesis and cofactor activity of FVIII. 8 Furthermore, VWF concentrates FVIII at the site of vascular injury. 9 The absence of VWF or the presence of defective VWF results in a secondary decrease of FVIII activity and a bleeding tendency known as von Willebrand disease. 7 Two major binding regions for VWF in the FVIII molecule have been reported, one within amino acid residues 1670 to 1689 in the amino-terminal region of LCh 10,11 and the other within residues 2303 to 2332 in the carboxy-terminal region of the C2 domain. 12,13 A3 fragments, composed of amino-terminal residues from 1672 to 1794, and LCh fragments lacking the amino-terminal acidic region of the C2 domain appear to have reduced affinities for VWF, indicating that both regions are essential for the high-affinity binding of VWF to FVIII. 14 The heavy chain is not directly involved in VWF interaction with FVIII. 15 Recently, Jacquemin et al reported that an anti-C1 monoclonal antibody (MoAb) inhibited FVIII binding to VWF, suggesting a possible role of C1 sequences in FVIII and VWF association. 16 Activation and inactivation of FVIII is regulated by VWF, and this regulatory mechanism appears to be essential for the maintenance of normal circulating levels of FVIII activity. Upon activation by thrombin, FVIII dissociates from VWF an...

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Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991, BLOOD102, 2002). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by <20%). To investigate the responsible residues in A2 domain for plasmin binding, the mutant forms of the A2 domain were expressed in baculovirus system and purified. Compared with wild type (23 nM), the affinity of R484A mutant was dramatically decreased by ~250-fold, and the affinities of K377A, K466A, R471A, and K523A mutants also were decreased by 10~40-folds, respectively. Especially, the addition of R484A mutant was reduced inactivation rate constant of plasmin-catalyzed FVIIIa by only ~40% of that of wild type. These findings demonstrate that Arg484 in the A2 domain contains plasmin-binding site responsible for plasmin-catalyzed FVIII(a) inactivation.

Summary Protein S functions as an activated protein C (APC)‐independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa‐dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17‐fold increase in Km for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in Km for factor X. Surface plasmon resonance‐based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (Kd; c. 10 nmol/l). Competition binding assays using Glu‐Gly‐Arg‐active‐site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg336 by factor IXa was c. 1·8‐fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down‐regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.

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Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmincatalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased ϳ2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was ϳ12-and ϳ3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A1 Factor VIII, a plasma protein deficient or defective in individuals with the severe congenital bleeding disorder hemophilia A, functions as a cofactor in the tenase complex, which is responsible for anionic phospholipid surface-dependent conversion of factor X to Xa by factor IXa (1). Factor VIII circulates as a complex with VWF 2 that protects and stabilizes the cofactor. Factor VIII is synthesized as a single chain molecule consisting of 2,332 amino acid residues with a molecular mass of ϳ300 kDa (2, 3). The factor VIII molecule can be divided into three domains arranged in the order of A1-A2-B-A3-C1-C2 according to the amino acid content homology. It is processed into a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B domain, linked to a light chain consisting of the A3, C1, and C2 domains (2-4). The catalytic efficacy of factor VIII in the tenase complex is enhanced over 10 5 times by conversion into an active form, factor VIIIa, by limited proteolysis by either thrombin or factor Xa (5). Both enzymes cleave factor VIII at Arg 372 and Arg 740 of the heavy chain and produce 50-kDa A1 and 40-kDa A2 subunits. The 80-kDa light chain is also cleaved at Arg 1689 generating a 70-kDa A3-C1-C2 subunit. Additionally, factor Xa cleaves at Arg 1721 and produces a 67-kDa A3-C1-C2 subunit. Proteolysis at Arg 372 and Arg 1689 is essential for generating factor VIIIa cofactor activity (6). Cleavage at the former site exposes a functional factor IXa-interactive site within the A2 domain that is cryptic in the unactivated molecule (7). Cleavage at the latter site liberates the cofactor from its carrier protein, VWF (8), contributing to the overall specific activity of the cofactor (9, 10).Factor VIIIa cofactor activity is down-regulated in the presence of serine proteases such as APC (5), factor Xa (5), and factor IXa (11) by proteolytic inactivation following cleavage at Arg 336 within the A1 subunit. This inactivation appears to be the result of altered interaction with the A2 subunit and an increased K m value of the truncated A1 for the substrate factor X (12, 13), the latter reaction reflecting loss of a fac...

We have recently reported that plasmin likely associates with the factor VIII light chain to proteolyze at Lys 36 within the A1 domain. In this study, we determined that the rate of plasmincatalyzed inactivation on the forms of factor VIIIa containing A1-( Factor VIII circulates as a complex with von Willebrand factor and functions as an essential cofactor in the tenase complex responsible for anionic phospholipid surface-dependent conversion of factor X to Xa by factor IXa (1). Molecular defects in factor VIII result in the congenital bleeding disorder, hemophilia A. Factor VIII is composed of 2,332 amino acid residues with a molecular mass of ϳ300 kDa and contains three types of structural domain, arranged in the order of A1-A2-B-A3-C1-C2 (2, 3). Mature factor VIII is processed to a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, together with heterogeneous fragments of a partially proteolyzed B domain, linked to a light chain consisting of the A3, C1, and C2 domains (2-4).Factor VIII is converted into an active form, factor VIIIa, by limited proteolysis catalyzed by either thrombin or factor Xa (5 is essential for generating factor VIIIa cofactor activity (6). Cleavage at the former site exposes a functional factor IXa-interactive site within the A2 domain that is cryptic in the unactivated molecule (7). Cleavage at the latter site liberates the cofactor from its carrier protein, von Willebrand factor (8), and contributes to the overall specific activity of the cofactor (9, 10). APC 2 (5), factor Xa (5), and factor IXa (11) are serine proteases that inactivate factor VIII(a) by cleavage at Arg 336 within the A1 subunit. This inactivation appears to be associated with an altered interaction between the A2 subunit and truncated A1 and is coupled with an increase in the K m value for the substrate, factor X (12, 13), reflecting loss of a factor X-interactive site within residues 337-372 (14). In addition, a second specific cleavage site for factor Xa, Lys 36 , was identified within the A1 subunit (13). Attack at this site also results in factor VIII inactivation mediated by an altered conformation of the A1 subunit limiting productive interaction with the A2 subunit (13).Plasmin is a potent fibrinolytic protease and is composed of a heavy chain consisting of five kringle domains and a light chain containing the catalytic domain. The protease associates with numerous proteins via the LBS on the exposed surface (15). Several reports have shown that plasmin proteolytically inacti-* This work was supported in part by Grant from MEXT KAKENHI 19591264 and Mitsubishi Pharma Research Foundation. An account of this work was presented at the 48th annual meeting of the American Society of Hematology, December 9, 2006, Orlando, FL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solel...