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Thromb Haemost 1998; 80(02): 302-309
DOI: 10.1055/s-0037-1615192
Rapid Communication
Schattauer GmbH

Purification of the Porcine Platelet GP IIb-IIIa Complex and the Propolypeptide of von Willebrand Factor

Teresa Royo
1   Cardiovascular Research Center, IIBBCSIC-Hospital de la Santa Creu i Sant Pau-UAB. Research Institute Sant Pau, Barcelona, Spain
,
Matilde Vidal
1   Cardiovascular Research Center, IIBBCSIC-Hospital de la Santa Creu i Sant Pau-UAB. Research Institute Sant Pau, Barcelona, Spain
,
Lina Badimon
1   Cardiovascular Research Center, IIBBCSIC-Hospital de la Santa Creu i Sant Pau-UAB. Research Institute Sant Pau, Barcelona, Spain
› Author AffiliationsSupported by FISss 92/0114, FISss 95/1381, Fundación de Investigación Cardiovascular (FIC)-Catalana Occidente and Spanish Atherosclerosis Foundation.
Further Information

Publication History

Received 03 July 1997

Accepted after resubmission 09 April 1998

Publication Date:
08 December 2017 (online)

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Summary

Platelet membrane glycoproteins (GP) are involved in platelet adhesion and aggregation. The glycoprotein IIb-IIIa complex (GP IIbIIIa) is a Ca2+-dependent heterodimer that binds fibrinogen and other adhesive proteins, thereby mediating platelet aggregation and adhesion. We have purified two major glycoproteins from pig platelets by Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl S-300 HR chromatography (Fitzgerald et al. Anal Biochem, 1985): i) the GP IIb-IIIa complex, GP IIb Mr = 140,000 and GP IIIa a single chain of Mr = 95,000-100,000; and ii) a predominant glycoprotein of high molecular weight, the propolypeptide of von Willebrand factor (Mr = 80,000-100,000). Western-blot analysis of the purified GP IIb-IIIa showed that only certain monoclonal antibodies against the human receptor specifically recognize the porcine complex. Differences between the porcine and human GP IIb-IIIa glycoproteins could partially explain the decreased inhibitory effects of GP IIb/IIIa-antagonists (against the human receptor) in porcine platelets.

 

  • References

  • 1 George JN, Nurden AT, Phillips DR. Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 1984; 311: 1084-98.
    CrossrefPubMedGoogle Scholar
  • 2 Kunicki TJ, Pidard D, Rosa JP, Nurden AT. The formation of Ca++-depen-dent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis. Blood 1981; 58: 268-78.
    PubMedGoogle Scholar
  • 3 Jennings LK, Phillips DR. Purification of glycoproteins IIb and IIIa from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-IIIa complex. J Biol Chem 1982; 257: 10458-66.
    PubMedGoogle Scholar
  • 4 Carrell NA, Fitzgerald LA, Steiner B, Erickson HP, Phillips DR. Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy. J Biol Chem 1985; 260: 1743-9.
    PubMedGoogle Scholar
  • 5 Phillips DR, Agin PP. Platelet membrane defects in Glanzmann’s thrombasthenia. Evidence for decreased amounts of two major glycoproteins. J Clin Invest 1977; 60: 535-45.
    CrossrefPubMedGoogle Scholar
  • 6 Levy-Toledano S, Tobelem G, Legrand C, Bredoux R, Degos L, Nurden A, Caen JP. Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function. Blood 1978; 51: 1065-71.
    PubMedGoogle Scholar
  • 7 Phillips DR, Jennings LK, Edwards HH. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol 1980; 86: 77-86.
    CrossrefPubMedGoogle Scholar
  • 8 Weiss HJ, Turitto VT, Baumgartner HR. Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules. Blood 1986; 67: 322-30.
    PubMedGoogle Scholar
  • 9 Lawrence JB, Gralnick HR. Monoclonal antibodies to the glycoprotein IIb-IIIa epitopes involved in adhesive protein binding: effects on platelet spreading and ultrastructure on human arterial subendothelium. J Lab Clin Med 1987; 109: 495-534.
    PubMedGoogle Scholar
  • 10 Ginsberg MH, Loftus JC, Plow EF. Cytoadhesins, integrins, and platelets. Thromb Haemost 1988; 59: 1-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Parise LV. The structure and function of platelet integrins. Curr Opin Cell Biol 1989; 1: 947-52.
    CrossrefPubMedGoogle Scholar
  • 12 Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992; 69: 11-25.
    CrossrefPubMedGoogle Scholar
  • 13 Ginsberg MK, Xiaoping D, O’Toole TE, Loftus JC, Plow EF. Platelet integrins. Thromb Haemost 1993; 70: 87-93.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Shattil SJ, Brass LF. Induction of the fibrinogen receptor on human platelets by intracellular mediators. J Biol Chem 1987; 262: 992-1000.
    PubMedGoogle Scholar
  • 15 Brass LF, Laposata M, Banga HS, Rittenhouse SE. Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi. J Biol Chem 1986; 261: 16838-47.
    PubMedGoogle Scholar
  • 16 Shattil SJ. Regulation of platelet anchorage and signaling by integrin αIIbβ3 . Thromb Haemost 1993; 70: 224-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein IIb/IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-14.
    PubMedGoogle Scholar
  • 18 Plow EF, Ginsberg MH. Cellular adhesion: GP IIb-IIIa as a prototypic adhesion receptor. Prog Hemost Thromb 1989; 9: 117-56.
    PubMedGoogle Scholar
  • 19 Furman MI, Gardner TM, Goldschmidt-Clermont PJ. Mechanisms of cyto-skeletal reorganization during platelet activation. Thromb Haemost 1993; 70: 229-32.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 20 Calvete JJ, Arias J, Alvarez MV, Lopez MM, Henschen A, González-Rodriguez J. Further studies on the topography of human platelet glycoprotein IIb: localization of monoclonal antibody epitopes and the putative glycoprotein IIIa- and fibrinogen-binding regions. Biochem J 1991; 273: 767-75.
    CrossrefPubMedGoogle Scholar
  • 21 Calvete JJ, Arias J, Alvarez MV, Lopez MM, Henschen A, González-Rodriguez J. Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa: localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites. Biochem J 1991; 274: 457-63.
    CrossrefPubMedGoogle Scholar
  • 22 González-Rodriguez J, Acuña AU, Alvarez MV, Jovin TM. Rotational mobility of the fibrinogen receptor glycoprotein IIb/IIIa or integrin αIIbβ3 in the plasma membrane of human platelets. Biochemistry 1994; 33: 266-74.
    CrossrefPubMedGoogle Scholar
  • 23 Fox JE, Phillips DR. Polymerization and organization of actin filaments within platelets. Semin Hematol 1983; 20: 243-60.
    PubMedGoogle Scholar
  • 24 Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.
    CrossrefPubMedGoogle Scholar
  • 25 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedGoogle Scholar
  • 26 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4.
    CrossrefPubMedGoogle Scholar
  • 27 Douvas AS, Stumph WE, Reyes P, Tan EM. Isolation and characterization of nuclear ribonucleoprotein complexes using human anti-nuclear ribonucleoprotein antibodies. J Biol Chem 1979; 254: 3608-16.
    PubMedGoogle Scholar
  • 28 Pueyo C, Badimon JJ, Royo T, Feigen LP, Badimon L. A mimetic of the RGDF-peptide [arginine-glycine-aspartic acid-phenylalanine] blocks aggregation and flow-induced platelet deposition on severely injured stenotic arterial wall. Effects on different animal models and in humans. Thromb Res 1996; 81: 101-12.
    CrossrefPubMedGoogle Scholar
  • 29 Fitzgerald LA, Leung B, Phillips DR. A method for purifying the platelet membrane glycoprotein IIb-IIIa complex. Anal Biochem 1985; 151: 169-77.
    CrossrefPubMedGoogle Scholar
  • 30 Stanton HC, Mersmann HJ. eds. Swine in cardiovascular research. Boca Raton, FL: CRC Press Inc.; 1986
    Google Scholar
  • 31 Niewiarowski S, Regoeczi E, Mustard JF. Platelet interaction with fibrinogen and fibrin: comparison of the interaction of platelets with that of fibro-blasts, leukocytes, and erythrocytes. Ann N Y Acad Sci 1972; 201: 72-83.
    CrossrefPubMedGoogle Scholar
  • 32 Thomas DP, Niewiarowski S, Ream VJ. Release of adenine nucleotides and platelet factor 4 from platelets of man and four other species. J Lab Clin Med 1970; 75: 607-18.
    PubMedGoogle Scholar
  • 33 Marguerie GA, Edgington TS, Plow EF. Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 1980; 255: 154-61.
    PubMedGoogle Scholar
  • 34 Hawiger J, Parkinson S, Timmons S. Prostacyclin inhibits mobilization of fibrinogen-binding sites on human ADP- and thrombin-treated platelets. Nature 1980; 283: 195-7.
    CrossrefPubMedGoogle Scholar
  • 35 Kornecki E, Niewiarowski S, Morinelli TA, Kloczewiak M. Effects of chymotrypsin and adenosine diphosphate on the exposure of fibrinogen receptors on normal human and Glanzmann’s thrombasthenic platelets. J Biol Chem 1981; 256: 5696-701.
    PubMedGoogle Scholar
  • 36 Meyers KM, Katz JB, Clemmons RM, Smith JB, Holmsen H. An evaluation of the arachidonate pathway of platelets from companion and food-producing animals, mink, and man. Thromb Res 1980; 20: 13-24.
    CrossrefPubMedGoogle Scholar
  • 37 Rucinski B, Niewiarowski S. Isolation and characterization of porcine platelets. Methods Enzymol 1989; 169: 22-6.
    PubMedGoogle Scholar
  • 38 Grinstein S, Furuya W. Comparative study of the major glycoproteins of the plasma membrane and secretory granule membranes of porcine platelets. Comp Biochem Physiol 1984; 78B: 657-62.
    PubMedGoogle Scholar
  • 39 Takami BH, Nichols WL, Kaese SE, Miller RS, Katzmann JA, Bowie EJW. Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa. Blood 1988; 72: 1740-7.
    PubMedGoogle Scholar
  • 40 Badimon L, Badimon JJ, Cohen M, Chesebro J, Fuster V. Thrombosis in stenotic and laminar flow conditions: effect of an antiplatelet GP IIb/IIIa monoclonal antibody fragment (7E3F(ab’)2 . Circulation 1989; 80 Suppl II-422 (abstract).
    PubMedGoogle Scholar
  • 41 Yasuda T, Gold HK, Fallon JT, Leinbach RC, Guerrero JL, Scudder LE, Kanke M, Shealy D, Rosa MJ, Collen D, Coller BS. Monoclonal antibody against the platelet glycoprotein (GP) IIb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs. J Clin Invest 1988; 81: 1284-91.
    CrossrefPubMedGoogle Scholar
  • 42 Coller BS, Folts JD, Smith SR, Scudder LE, Jordan R. Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GP IIb/IIIa receptor: correlation with bleeding time, platelet aggregation and blockade of GP IIb/IIIa receptors. Circulation 1989; 80: 1766-74.
    CrossrefPubMedGoogle Scholar
  • 43 Cook NS, Bruttger O, Pally C, Hagenbach A. The effects of two synthetic glycoprotein IIb/IIIa antagonists, Ro 43-8857 and L-700,462, on platelet aggregation and bleeding in guinea-pigs and dogs: evidence that Ro 43-8857 is orally active. Thromb Haemost 1993; 70: 838-47.
    Article in Thieme ConnectPubMedGoogle Scholar