Combinatorial peptides directed to inhibitory antibodies against human blood clotting factor VIII.
Eva-Maria Kopecky, Sabine Greinstetter, Ingrid Pabinger, Andrea Buchacher, Jürgen Römisch, Alois Jungbauer
Thrombosis and haemostasis
94
933-41
2004
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The development of antibodies against blood clotting factor VIII is a major complication affecting 20-30% of hemophilia A patients receiving replacement with FVIII concentrates.This study investigated generating peptides acting as broadly neutralizing agents to block factor VIII antibodies.These peptides were selected from dual positional scanning decapeptide libraries on cellulose membranes. From this library comprising 6.8 x 10(12) peptides we selected 468 peptides for further screening rounds. Finally we identified two decapeptides with the ability to block 8 out of 10 inhibitory antibodies from sera of patients with FVIII inhibitors demonstrated by competition assays. Sequence alignment of the peptides showed similarity with several domains in the FVIII molecule demonstrating the mimotope nature of the selected peptides. Our results show the efficiency of the combinatorial library approach and show the potential of combinatorial peptides to compete out polyclonal inhibitor IgG from a broad range of patients' sera. Combinatorial peptides could be novel and highly effective drug candidates for alternative treatment in patients with factor VIII inhibitors. | 16363233
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Binding of factor VIII inhibitors to discrete regions of the factor VIII C2 domain disrupt phospholipid binding.
Deborah A Lewis, Karen D Moore, Thomas L Ortel
Blood coagulation fibrinolysis : an international journal in haemostasis and thrombosis
14
361-8
2003
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We characterized seven factor VIII inhibitors with epitopes in the C2 domain of factor VIII using a series of factor V C2 domain chimeras that substituted exon-sized fragments of the C2 domain of factor VIII for the corresponding regions of factor V. All inhibited co-factor activity of factor VIII and six inhibited binding of factor VIII to phosphatidylserine. Inhibitors Hz, JN and GK32 bound epitopes within amino acids S2173-K2281; inhibitors GK24 and TO bound epitopes within amino acids V2223-Y2332; and inhibitors UNC11 and UNC12 bound epitopes throughout the C2 domain (amino acids S2173-Y2332). Inhibitors Hz, JN and UNC12 inhibited the co-factor activity of chimera 5A, which substituted amino acids S2173-Q2222 of factor VIII for the corresponding region of factor V, in a prothrombinase assay. This inhibition could be partially reversed by pre-incubation of chimera 5A with phospholipid vesicles, suggesting that these antibodies interfered with phospholipid binding. Inhibitors UNC11 and UNC12, on the other hand, did not inhibit the binding of chimera 1 A to phosphatidylserine, suggesting that binding to the segment spanning amino acids V2282-Y2332 does not necessarily block phospholipid binding. These results agree with the model of the phospholipid-binding site determined by crystal structure of the C2 domain of factor VIII. | 12945878
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