Identification of Novel Peptide Antagonists for von Willebrand Factor Binding to the Platelet Glycoprotein Ib Receptor from a Phage Epitope Library

Summary

We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH₂-terminal region of the phage gene III protein. This library, containing over 10⁷different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein lb interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

• References

• 1 Lenstra JA, Erkens JH F, Langeveld JG A, Posthumus WP A, Meloen RH, Gebauer F, Correa I, Enjuanes L, Stanley KK. Isolation of sequences from a random-sequence expression library that mimic viral epitopes. J Immunol Methods 1992; 152: 149-157
• 2 Cwirla SE, Peters EA, Barrett RW, Dower WJ. Peptides on phage: A vast library of peptides for identifying ligands. Proc Natl Acad Sci 1990; 87: 6378-6382
• 3 Devlin JJ, Panganiban LC, Devlin PE. Random peptide libraries: a source of specific protein binding molecules. Science 1990; 249: 404-406
• 4 Felici F, Castagnoli L, Musacchio A, Japelli R, Cesareni G. Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J Mol Biol 1991; 222: 301-310
• 5 Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 1990; 249: 386-390
• 6 Geysen HM, Rodda SJ, Mason TJ, Tribbick G, Shoofs PG. Strategies for epitope analysis using peptide synthesis. J Immunol Methods 1987; 102: 259-274
• 7 Fodor SP A, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable parallel chemical synthesis. Science 1991; 251: 767-772
• 8 Houghten RA, Pinella C, Blondelle SE, Appel JR, Dooley CT, Cuervo JH. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 1991; 354: 84-86
• 9 Houghten RA, Appel JR, Blondelle SE, Cuervo JH, Dooley CT, Pinilla C. The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides. BioTechniques 1992; 13: 412-421
• 10 Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991; 354: 82-84
• 11 Stephen CW, Lane DP. Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library J Mol Biol 1992; 225: 577-583
• 12 Luzzago A, Felici F, Tramontano A, Pessi A, Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides. I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Gene 1993; 128: 51-57
• 13 Oldenburg KR, Loganathan D, Goldstein IJ, Schultz PG, Gallop MA. Peptide ligands for a sugar-binding protein isolated from a random peptide library USA. Proc Natl Acad Sci 1992; 89: 5393-5397
• 14 Scott JK, Loganathan D, Easley RB, Gong X, Goldstein IJ. A family of concanavalin A-binding peptides from a hexapeptide library. Proc Natl Acad Sci USA 1992; 89: 5398-5402
• 15 O'Neil KT, Hoess RH, Jackson SA, Ramachandran NS, Mousa SA, DeGrado WF. Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage epitope library. Proteins: Structure, Function, and Genetics 1992; 14: 509-515
• 16 Koivunen E, Gay DA, Ruoslahti E. Selection of peptides binding to the α5β1 integrin from phage display library. J Biol Chem 1993; 268: 20205-20210
• 17 Smith GP, Schultz DA, Ladbury JE. A ribonuclease S-peptide antagonist discovered with a bacteriophage display library. Gene 1993; 128: 37-42
• 18 Blond-Elguindi S, Cwirla SE, Dower WJ, Lipshutz RJ, Sprang SR, Sambrook JF, Gething J-JH. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 1993; 75: 717-728
• 19 Dedman JR, Kaetzel MA, Chan HC, Nelson DJ, Jamieson GA. Selection of targeted biological modifiers from a bacteriophage library of random peptides. The identification of novel calmodulin regulatory peptides J Biol Chem 1993; 268: 23025-23030
• 20 Stel HV, Sakariassen KS, deGroot PG, van Mourik JA, Sixma JJ. Von Willebrand factor in the vessel wall mediates platelet adherence. Blood 1985; 65: 85-90
• 21 Weiss HJ, Turitto VT, Baumgartner HR. Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, and storage granules. Blood 1986; 67: 322-330
• 22 Baumgartner HR, Tschopp TB, Weiss HJ. Defective adhesion of platelets to subendothelium in von Willebrand’s disease and Bemard-Soulier syndrome. Thromb Haemost 1978; 39: 782-783
• 23 Pareti FI, Fujimura Y, Dent JA, Holland LZ, Zimmerman TS, Ruggeri ZM. Isolation and characterization of a collagen binding domain in human von Willebrand factor. J Biol Chem 1986; 261: 15310-15315
• 24 Pareti FI, Niiya K, McPherson JM, Ruggeri ZM. Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III. J Biol Chem 1987; 262: 13835-13841
• 25 Fujimura Y, Titani K, Holland LZ, Roberts JR, Kostel P, Ruggeri ZM, Zimmerman TS. A heparin-binding domain of human von Willebrand factor. Characterization and localization to a tryptic fragment extending from amino acid residue Val-449 to Lys-728 J Biol Chem 1987; 262: 1734-1739
• 26 Weiss HJ, Turitto VT, Baumgartner HR. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. I. Shear rate-dependent decrease of adhesion in von Willebrand’s disease and the Bemard-Soulier syndrome. J Lab Clin Med 1978; 92: 750-764
• 27 Fuster W, Bowie EJ, Lewis JC, Fass DN, Owen CA, Brown AL. Resistance to arteriosclerosis in pigs with von Willebrand’s disease. Spontaneous and high cholesterol diet-induced arteriosclerosis. J Clin Invest 1978; 61: 722-730
• 28 Nichols TC, Bellinger DA, Johnson TA, Lamb MA, Griggs TR. von Willebrand’s disease prevents occlusive thrombosis in stenosed and injured porcine coronary arteries. Circulation Research 1986; 59: 15-26
• 29 Bellinger DA, Nichols TC, Read MS, Reddick RL, Lamb MA, Brinkhous KM, Evatt BL, Griggs TR. Prevention of occlusive coronary artery thrombosis by a murine monoconal antibody to porcine von Willebrand factor. Proc Natl Acad Sci USA 1987; 84: 8100-8104
• 30 Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffman T. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest 1989; 83: 288-297
• 31 Fujimura Y, Titani K, Holland LZ, Russel SR, Roberts JR, Elder JH, Ruggeri ZM, Zimmerman TS. von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib J Biol Chem 1986; 261: 381-385
• 32 Mohri H, Fujimura Y, Shima M, Yoshioka A, Houghten RA, Ruggeri ZM, Zimmerman TS. Structure of the von Willebrand factor domain interacting with glycoprotein Ib. J Biol Chem 1988; 263: 17901-17904
• 33 Mohri H, Yoshioka A, Zimmerman TS, Ruggeri ZM. Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites. J Biol Chem 1989; 264: 17361-17367
• 34 Berndt MC, Ward CM, Booth WJ, Castaldi PA, Mazurov AV, Andrews RK. Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin Biochemistry 1992; 31: 11144-11151
• 35 Rabinowitz I, Randi AM, Shindler KS, Tuley EA, Rustagi PK, Sadler JE. Type IIB Mutation His-505-Asp Implicates a New Segment in the Control of von Willebrand Factor Binding to Platelet Glycoprotein Ib. J Biol Chem 1993; 268: 20497-20501
• 36 Coller BS, Gralnick HR. Studies on the mechanism of ristocetin-induced platelet agglutination: Effects of structural modification of ristocetin and vancomycin. J Clin Invest 1977; 302-312
• 37 Sugimoto M, Mohri H, McClintock RA, Ruggeri ZM. Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin. A model for the regulation of von Willebrand factor binding to platelet glycoprotein Ib J Biol Chem 1991; 266: 18172-18178
• 38 Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979; 254: 5357-5363
• 39 Allain JP, Cooper HA, Wagner RH, Brinkhous KM. Platelets fixed with paraformaldehyde: A new reagent for assay of von Willebrand factor and platelet aggregation factor. J Lab Clin Med 1975; 85: 319-328
• 40 Sugimoto M, Ricca G, Hrinda ME, Schreiber AB, Searfoss GH, Bottini E, Ruggeri ZM. Functional modulation of the isolated glycoprotein lb binding domain of von Willebrand factor expressed in Escherichia coli. Biochemistry 1991; 30: 5202-5209
• 41 Fields CG, Lloyd DH, MacDonald RL, Otteson KM, Noble RL. HBTU Activation for automated Fmoc solid-phase peptide synthesis. Peptide Research 1991; 4: 95-101
• 42 King DS, Fields CG, Fields GB. A cleavage method which minimized side-reactions following Fmoc solid phase peptide synthesis. Int J Peptide Protein Res 1990; 36: 255-266
• 43 Parmley SF, Smith GP. Antibody-selectable filamentous fd phage vectors: Affinity purification of target genes. Gene 1988; 73: 305-318
• 44 Prior C, Chu V, Holt J, Windisch V, Lee T, Mitschelen J, Newman J, Ricca G, Tarr C, Hrinda M. Case study in process development/Production and functional characterization of a recombinant fragment of von Willebrand factor(vWF): An antagonist to platelet receptor GPIb. Biotechnology 1992; 10: 66-73
• 45 Prior CP, Chu V, Cambou B, Dent JA, Ebert B, Gore R, Holt J, Irish T, Lee T, Mitschelen J, McClintock RA, Searfoss G, Ricca GA, Tarr C, Weber D, Ware JL, Ruggeri ZM, Hrinda M. Optimization of a recombinant von Willebrand factor fragment as an antagonist of the platelet glycoprotein Ib receptor. Biotechnology 1993; 11: 709-713
• 46 Gralnick HR, Williams S, McKeown L, Kramer W, Krutzsch H, Gorecki M, Pinet A, Garfinkel LI. A monomeric von Willebrand factor fragment, Leu-504-Ser-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX. Proc Natl Acad Sei USA 192 89: 7880-7884
• 47 Ruggeri ZM, Ware J. von Willebrand Factor. The FASEB Journal 1993; 7: 308-316
• 48 Bass S, Greene R, Wells JA. Hormone Phage: An enrichment method for variant proteins with altered binding properties. Proteins: Structure, Function and Genetics 1990; 8: 309-314
• 49 Barrett RW, Cwirla SE, Ackerman MS, Olson AM, Peters EA, Dower WJ. Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage. Anal Biochem 1992; 204: 357-364