Midori Shima

Role of factor VIII C2 domain in factor VIII binding to factor Xa

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248–2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K d ) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nm, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248–2285) demonstrated that a peptide designated EP-2 (residues 2253–2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.

Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689

Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K d values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and theK d values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC50, 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)′2 of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg1689. Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg1689.

Role of Activation of the Coagulation Factor VIII in Interaction with vWf, Phospholipid, and Functioning within the Factor Xase Complex

Blood coagulation factor VIII (fVIII) in its nonactivated form circulates in plasma in a complex with von Willebrand factor (vWf). Upon activation by thrombin- or factor Xa-mediated site-specific proteolysis, activated fVIII (fVIIIa) serves as a cofactor for factor IXa. This protein complex assembled on a phospholipid surface (factor Xase) activates factor X. This complex plays the key role in the intrinsic pathway of blood coagulation. We reviewed the molecular events triggered by fVIII activation, which are required for the assembly and functioning of the Xase complex, including fVIIIa dissociation from vWf and a significant increase of fVIII affinity for binding to the phospholipid surface. Both events are mediated by activation-related cleavage within fVIII light chain (LCh), releasing the 40 amino-acid N-terminal LCh peptide, which is followed by a conformational change within the C2 domain. The conformational change within LCh is also required for the optimal fVIII cofactor functioning within the factor Xase complex, exerted via fVIIIa interactions with phospholipid, factor IXa, and factor X. Since factor IXa not only stabilizes but also proteolytically inactivates fVIIIa within the factor Xase complex, the stability of the membrane-bound fVIIIa in the presence and absence of factor IXa is discussed. In conclusion, we outline some new possible directions of the research. One of them arises from the recently demonstrated ability of plasma lipoproteins to provide a phospholipid surface for the assembly of the factor Xase complex in vitro. This finding raises a possibility that lipoproteins participate in factor Xase functioning in vivo and suggests a direct link between elevated levels of lipoproteins associated with atherosclerosis and increased thrombogenicity associated with this disease.

Use of surface plasmon resonance for studies of protein-protein and protein-phospholipid membrane interactions. Application to the binding of factor VIII to von Willebrand factor and to phosphatidylserine-containing membranes.

The surface plasmon resonance phenomenon is used for real time measurements of protein-protein and protein-membrane interactions. In the present study two surface plasmon resonance-based binding assays permitting study of the interaction of coagulation factor VIII (fVIII) with von Willebrand factor (vWf) and phospholipid have been developed. These interactions of fVIII are required for maintenance of fVIII concentration in circulation and for the assembly of the functional factor Xase complex, respectively. With these binding assays, the role of the light chain (LCh) in fVIII binding to vWf and to immobilized phospholipid monolayers and intact vesicles containing 25% phosphatidylserine (PS) and 4% PS was examined. The finding that Kd of LCh binding to vWf (3.8 nM) is 9.5 times higher than that of fVIII (0.4 nM), indicates that the heavy chain (HCh) is required for the maximal affinity of fVIII for vWf. In contrast, affinities of LCh for 25/75 PS/phosphatidylcholine (PC) monolayers and 4/76/20 PSPC-phosphatidylethanolamine (PE) vesicles are similar to that of fVIII, indicating that LCh is solely responsible for these interactions. It was also examined how removal of the acidic region affects the binding affinity of the remaining part of LCh for vWf and phospholipid. It was demonstrated that the loss of the LCh acidic region upon thrombin cleavage leads to an 11 and 160-fold increase in the dissociation rate constant (k(off) value) and a 165 and 1500-fold increase in the Kd value of the binding of fVIII fragment A3-C1-C2 to vWf compared to that of LCh and fVIII, respectively. In contrast, the binding affinity of A3-C1-C2 for PS-containing membranes was 8-11-fold higher than that of LCh. Possible conformational change(s) in C2 domain upon removal of the acidic region were studied using anti-fVIII monoclonal antibody NMC-VIII/5 with an epitope within the C2 domain of LCh as a probe. The determined lower binding affinity of A3-C1-C2 for NMC-VIII/5 immobilized to a sensor chip than that of LCh, indicates that these conformational changes do occur.

Mapping of the minimal domain encoding a conformational epitope by λ phage surface display : factor VIII inhibitor antibodies from haemophilia A patients

Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies, resulting in reduced efficacy of the therapy. Determination of fVIII epitopes for the alloantibodies is essential for an understanding of their inhibitory effect on blood coagulation. Random fragments of fVIII displayed on lambda phage particles were selected using two patient plasmas immobilized onto the surface of a microtiter plate. A set of clones defined the minimal domain that consisted of 157 amino acid residues including cysteine at both boundaries. The minimal domain absorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant for the plasmas. Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed by the disulfide bridge was recognized by the antibodies. The epitope domain defined overlaps with fVIII binding sites to vWf and phospholipid, and may play an important role in blood coagulation. Thus, the bacteriophage lambda surface display may be useful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope.

Identification of a factor VIII peptide, residues 2315-2330, which neutralizes human factor VIII C2 inhibitor alloantibodies: requirement of Cys2326 and Glu2327 for maximum effect.

Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3‐C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy‐terminal region of the C2 domain (residues 2288–2332). We found one peptide (2315–2330, L9) which neutralized the anti‐FVIII activity of four out of five different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173–2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218–2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315–2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg2320 and Cys2326 or Glu2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.

Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates

Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin-agarose in a reversible manner. The K(d) values for factor VIII, fibrinogen, factor XIII and protein C were 1.2x10(-8), 4.4x10(-8), 2.8x10(-7) and 8.1x10(-5) M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (k(ass)) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.

The Role of Platelet Von Willebrand Factor in the Binding of Factor VIII to Activated Platelets

Factor VIII binds to activated platelets and contributes to the tenase complex assembled on the platelet membrane surface. We have examined the role of platelet von Willebrand factor in the binding of factor VIII to platelets using a platelet captured enzyme-linked immunosorbent assay. Purified factor VIII bound to activated normal platelets in a dose dependent manner. Factor VIII also bound to platelets obtained from a patient with Type 2N von Willebrand disease, although in this case the binding was reduced to approximately 50% of that seen with control platelets. Furthermore, factor VIII bound to Type 3 von Willebrand disease platelets in the absence of detectable von Willebrand factor. In this instance the binding reaction appeared to be approximately 30% of that seen with the same number of normal platelets. An anti-A3 domain monoclonal antibody, NMC-VIII/10, which recognizes the amino-terminal acidic region of the factor VIII light chain, and an anti-C2 domain monoclonal antibody, NMC-VIII/5, which also moderates the binding of factor VIII to phosphatidylserine, inhibited the association between factor VIII and platelets. Inhibition was more remarkable with NMC-VIII/5 than with NMC-VIII/ 10 but not complete. The findings suggest that the binding of factor VIII to activated platelets is not based on a single ligand-receptor relationship, although a predominant role exists for the platelet von Willebrand factor. Furthermore, both the amino-terminal acidic region of the A3 domain and the C2 domain participate in the binding of factor VIII to activated platelets.