Neutralization of the Antiheparin Activity of Platelet Factor 4 by a Monoclonal Antibody

Summary

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4’s ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/ PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

• References

• 1 Lane DA. Heparin binding and neutralizing proteins. In: Heparin. Chemical and Biological Properties, Clinical Management. Lane DA, Lindahl U. (eds) Edward Arnold; London: 1989. pp 363-391
• 2 Handin RI, Cohen HJ. Purification and binding properties of human platelet factor four. J Biol Chem 1976; 251: 4273-4282
• 3 Levine SP, Wohl HF. Human platelet factor 4: purification and characterization by affinity chromatography. Purification of human platelet factor 4. J Biol Chem 1976; 251: 324-328
• 4 Deuel TF, Keim PS, Farmer M, Heinrikson RL. Amino acid sequence of human platelet factor 4. Proc Natl Acad Sei USA 1977; 74: 2256-2258
• 5 Ginsberg MH, Hoskins R, Sigrist P, Painter RG. Purification of a heparin-neutralizing protein from rabbit platelets and its homology with human platelet factor 4. J Biol Chem 1979; 254: 12365-12371
• 6 McManus LM, Morely CA, Levine SP, Pinckard RN. Platelet activating factor (PAF) induce release of platelet factor 4 (PF4) in vitro and during IgE anaphylaxis in the rabbit. J Immunol 1979; 123: 2835-2841
• 7 Ciaglowski RE, Snow J, Walz DA. Isolation and aminoacid sequence of bovine platelet factor 4. Arch Biochem Biophys 1986; 250: 249-256
• 8 Rucinski B, Poggi A, James P, Holt JC, Niewiarowski S. Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted proteins. Blood 1983; 61: 1072-1080
• 9 Eisman R, Surrey S, Ramachendran B, Schwartz E, Poncz M. Structural and functional comparison of the genes for human platelet factor 4 and PF4_alt . Blood 1990; 76: 336-344
• 10 Doi T, Greenberg SH, Rosenberg RD. Structure of the rat platelet factor 4 gene: a marker for megakaryocyte differentiation. Mol Cell Biol 1987; 7: 898-904
• 11 Barone AD, Ghrayeb J, Hammerling U, Zucker MB, Thorbecke GJ. The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity. J Biol Chem 1988; 263: 8710-8715
• 12 Park KS, Rifat S, Eck H, Adachi K, Surrey S, Poncz M. Biological and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein. Blood 1990; 75: 1290-1295
• 13 Loscalzo J, Melnick B, Handin RI. The interaction of platelet factor four and glycosaminoglycans. Arch Biochem Biophys 1985; 240: 446-455
• 14 Cowan S, Bakshi EN, Machin KJ, Isaacs NW. Binding of heparin to human platelet factor 4. Biochem J 1986; 234: 485-488
• 15 Denton J, Lane DA, Thunberg L, Slater AM, Lindahl U. Binding of platelet factor 4 to heparin oligosaccharides. Biochem J 1983; 209: 455-460
• 16 Lane DA, Denton J, Flynn AM, Thunberg L, Lindahl U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem J 1984; 218: 725-732
• 17 Mantovani A. Tumor-associated macrophages. Curr Opin Immunol 1990; 2: 689-692
• 18 Begg GS, Pepper DS, Chesterman CN, Morgan FJ. Complete covalent structure of human p-thromboglobulin. Biochemistry 1978; 17: 1739-1744
• 19 Rybak ME, Gimbrone MA, Davies PF, Handin RI. Interaction of platelet factor four with cultured vascular endothelial cells. Blood 1989; 73: 1534-1539
• 20 Rao AK, Niewiarowski S, James P, Holt JC, Harris M, Elfenbein B, Bastl C. Effect of heparin on the in vivo release and clearance of human platelet factor 4. Blood 1983; 61: 1208-1214
• 21 Celia G, Colby SI, Taylor AD, McCracken L, Parisi AF, Sasahara AA. Platelet factor 4 (PF4) and heparin-released platelet factor 4 (HR-PF4) in patients with cardiovascular disorders. Thromb Res 1983; 29: 499-509
• 22 Prosdocimi M, Scattolo N, Zatta A, Fabris F, Stevanato F, Girolami A, Celia G. Clearance and in vivo release by heparin of human platelet factor 4 (PF4) in the rabbit. Thromb Haemostas 1984; 52: 157-159
• 23 Lawler JW. Prediction of the secondary structure of platelet factor 4 and P-thromboglobulin from their amino acid sequences. Thromb Res 1981; 21: 121-127
• 24 Charles RSt, Walz DA, Edwards FP. The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution. J Biol Chem 1989; 264: 2092-2099
• 25 Zatta A, Zanetti A, Dejana E, Prosdocimi M. At low extracellular calcium concentration platelet activating factor induces beta-throm-boglobulin release from human platelets through thromboxaneendoperoxides formation. Agents Actions 1987; 22: 151-158
• 26 Medici L, Di Martino A, Celia G, Callegaro L, Prosdocimi M. Improved method for purification of human platelet factor 4 by affinity and ion-exchange chromatography. Thromb Res 1989; 54: 277-287
• 27 Galfre G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Meth Enzymol 1981; 73: 3-46
• 28 McKinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods 1987; 96: 271-278
• 29 Parham P. Preparation and purification of active fragments from mouse monoclonal antibodies. In: Handbook of Experimental Immunology. Weir DM. (ed). Vol. 1 Blackwell Scientific Publisher; Oxford: 1986. pp 14.1-23
• 30 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76-85
• 31 Saggin L, Ausoni S, Gorza L, Sartore S, Schiaffino S. Troponin T switching in developing rat heart. J Biol Chem 1988; 263: 18488-18492
• 32 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 33 Towbin H, Staehlin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
• 34 Saggin L, Gorza L, Ausoni S, Schiaffino S. Troponin I switching in the developing heart. J Biol Chem 1989; 264: 16299-16302
• 35 Kinlough-Rathbone RL, Packham MA, Mustard JF. Platelet aggregation. In: Methods in Hematology. Measurements of Platelet Function. Harker LA, Zimmerman TS. (eds). Churchill & Livingstone; Edinburgh: 1983. pp 64-91
• 36 Aitken A, Geisow MJ, Fiondlay JBC, Holmes C, Yarwood A. Peptide preparation and characterization. In: Protein Sequencing. A Practical Approach. Findlay JBC, Geisow MJ. (eds). IRL Press; Oxford: 1989. pp 43-68
• 37 Ashbridge KR, Prestidge RL, Booth RJ, Watson JD. The mapping of an antibody-binding region on the Mycobacterium tubercolosis 19 kilodalton antigen. J Immunol 1990; 144: 3137-3142
• 38 Eberle AN, Atherton E, Dryland A, Sheppard RC. Peptide synthesis. Part 9, solid phase synthesis of melanin concentrating hormone using a continuous flow polyamide method. Chem Soc Perkin Trans 1986; 1: 361-367
• 39 Holt JC, Harris ME, Holt AM, Lange E, Henshen A, Niewiarowski S. Characterization of human platelet basic protein, a precursor form of low affinity platelet factor 4 and p-thromboglobulin. Biochemistry 1986; 25: 1988-1996
• 40 Berzofsky JA. Intrinsic and extrinsic factors in protein antigenic structure. Science 1985; 229: 932-940
• 41 Ciaglowski RE, Martinez J, Ryan TJ, Lewandowski LJ, Barnhart MI, Weller MA, Chen S, Walz DA. Monoclonal antibodies to bovine platelet factor 4: species interactions to platelets and megakaryocytes using indirect immunocytofluorescence. Thromb Res 1986; 41: 855-865
• 42 Barber AJ, Kaser-Glanzmann R, Jakabova M, Lusher EF. Characterization of chondroitin 4-sulphate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets. Biochem Biophys Acta 1972; 286: 312-329
• 43 Huang SS, Huang JS, Deuel TF. Proteoglycan carrier of human platelet factor 4. J Biol Chem 1982; 257: 11546-11550
• 44 Levine SP, Knieriem LK, Rage MA. Platelet factor 4 and platelet secreted proteoglycan: immunologic characterization by crossed immunoelectrophoresis. Blood 1990; 75: 902-910
• 45 Rucinski B, Niewiarowski S, Strzyzewski M, Holt JC, Mayo KH. Human platelet factor 4 and its C-terminal peptides: heparin binding and clearance from the circulation. Thromb Haemostas 1990; 63: 493-498
• 46 Rucinski B, Niewiarowski S, Strzyzewski M, Holt JC, Mayo K. Structural requirements for the expression of heparin binding activity of human platelet factor 4 (PF4) and its clearance from the circulation. Blood 1988; 72: 1255 (Abstr)
• 47 Marcum JA, McKenney JB, Rosenberg RD. Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparinlike molecules bound to endothelium. J Clin Invest 1984; 74: 341-350
• 48 Maione TE, Gray GS, Petro J, Hunt AJ, Donner AL, Bauer SI, Carson HF, Sharpe RJ. Inhibition of angiogenesis by recombinant human platelet factor 4 and related peptides. Science 1990; 247: 77-79
• 49 Zucker MB, Katz IR, Thorbecke GJ, Milot DC, Holt J. Immunoregulatory activity of peptides related to platelet factor 4. Proc Natl Acad Sci USA 1989; 86: 7571-7574
• 50 Maione TE, Gray GS, Hunt AJ, Sharpe RJ. Inhibition of tumor growth in mice by an analogue of Platelet Factor 4 that lacks affinity for heparin and retains potent angiostatic activity. Cancer Res 1991; 51: 2077-2083