Download PDF
Thromb Haemost 1992; 67(01): 137-143
DOI: 10.1055/s-0038-1648395
Original Articles
Schattauer GmbH Stuttgart

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

Leopoldo Saggin
1   The Dept. of Vascular Biology, FIDIA S.p.A., Abano Terme, Italy
,
Flavia Cazzola
2   Advanced Technology Division , FIDIA S.p.A., Abano Terme, Italy
,
Giuseppe Corona
2   Advanced Technology Division , FIDIA S.p.A., Abano Terme, Italy
,
Emanuela Salvatico
1   The Dept. of Vascular Biology, FIDIA S.p.A., Abano Terme, Italy
,
Giuseppe Cella
3   II Chair of Medicine, Institute of Medical Semeiotics, University of Padua, Padua, ltaly
,
Marco Prosdocimi
1   The Dept. of Vascular Biology, FIDIA S.p.A., Abano Terme, Italy
› Author Affiliations
Further Information

Publication History

Received 15 May 1991

Accepted after revision 11 July 1991

Publication Date:
02 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

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
    Google Scholar
  • 2 Handin RI, Cohen HJ. Purification and binding properties of human platelet factor four. J Biol Chem 1976; 251: 4273-4282
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 7 Ciaglowski RE, Snow J, Walz DA. Isolation and aminoacid sequence of bovine platelet factor 4. Arch Biochem Biophys 1986; 250: 249-256
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 9 Eisman R, Surrey S, Ramachendran B, Schwartz E, Poncz M. Structural and functional comparison of the genes for human platelet factor 4 and PF4alt . Blood 1990; 76: 336-344
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 13 Loscalzo J, Melnick B, Handin RI. The interaction of platelet factor four and glycosaminoglycans. Arch Biochem Biophys 1985; 240: 446-455
    CrossrefPubMedGoogle Scholar
  • 14 Cowan S, Bakshi EN, Machin KJ, Isaacs NW. Binding of heparin to human platelet factor 4. Biochem J 1986; 234: 485-488
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 17 Mantovani A. Tumor-associated macrophages. Curr Opin Immunol 1990; 2: 689-692
    CrossrefPubMedGoogle Scholar
  • 18 Begg GS, Pepper DS, Chesterman CN, Morgan FJ. Complete covalent structure of human p-thromboglobulin. Biochemistry 1978; 17: 1739-1744
    CrossrefPubMedGoogle Scholar
  • 19 Rybak ME, Gimbrone MA, Davies PF, Handin RI. Interaction of platelet factor four with cultured vascular endothelial cells. Blood 1989; 73: 1534-1539
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 27 Galfre G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Meth Enzymol 1981; 73: 3-46
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    Google Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 32 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 34 Saggin L, Gorza L, Ausoni S, Schiaffino S. Troponin I switching in the developing heart. J Biol Chem 1989; 264: 16299-16302
    PubMedGoogle Scholar
  • 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
    Google Scholar
  • 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
    Google Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 40 Berzofsky JA. Intrinsic and extrinsic factors in protein antigenic structure. Science 1985; 229: 932-940
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 43 Huang SS, Huang JS, Deuel TF. Proteoglycan carrier of human platelet factor 4. J Biol Chem 1982; 257: 11546-11550
    PubMedGoogle Scholar
  • 44 Levine SP, Knieriem LK, Rage MA. Platelet factor 4 and platelet secreted proteoglycan: immunologic characterization by crossed immunoelectrophoresis. Blood 1990; 75: 902-910
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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)
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar