A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649 -1689, also controls fVIII⅐vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII⅐vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14-and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K d 72 nM) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K d 564 -660 nM) compared with LCh (K d 3.8 nM). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170 -2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf.
The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.
SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.
The anti-factor VIII (fVIII) C2 domain monoclonal antibody ESH8 inhibits fVIII activity only when fVIII is bound to von Willebrand factor (vWf). However, ESH8 binds with similar affinity to fVIII and fVIII⅐vWf complex, and it does not affect the kinetics of thrombin cleavage at positions 372 and 740 within the fVIII heavy chain and at 1689 within the light chain. The latter is required for fVIII release from vWf. We showed that ESH8 reduced the initial rate of thrombin-activated fVIII (fVIIIa) release from vWf by 4.3-fold compared to that in the absence of antibody. The complex of vWf⅐fVIII⅐ESH8 was activated, and the rate constant determined for fVIIIa dissociation from vWf was 4 ؋ 10. We constructed a mathematical model incorporating the measured rates for fVIIIa release from vWf and for inactivation of heterotrimeric fVIIIa due to the spontaneous loss of the A2 subunit and found that the decreased release rate is sufficient to explain our experimentally observed inhibition of fVIII activity by ESH8. We hypothesize that the slowed rate of fVIIIa release from vWf in the presence of ESH8 allows time for inactivation of unstable fVIIIa prior its participation in the formation of the factor Xase complex. The relevance of these findings is illustrated by our observation that reduction of fVIIIa release from vWf represents an additional mechanism of fVIII inhibition by an anti-C2 domain antibody (epitope 2218 -2307) from a hemophilia A patient. This rare antibody binds to a more amino-terminal epitope than other human anti-C2 inhibitors, resulting in its lack of inhibition of fVIII binding to vWf but not to phospholipid. These two fVIII ligands therefore bind to C2 sites which do not overlap completely.
Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.
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