Purification of the Porcine Platelet GP IIb-IIIa Complex and the Propolypeptide of von Willebrand FactorSupported by FISss 92/0114, FISss 95/1381, Fundación de Investigación Cardiovascular (FIC)-Catalana Occidente and Spanish Atherosclerosis Foundation.
03 July 1997
Accepted after resubmission 09 April 1998
08 December 2017 (online)
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 7 Phillips DR, Jennings LK, Edwards HH. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol 1980; 86: 77-86.
- 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.
- 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.
- 10 Ginsberg MH, Loftus JC, Plow EF. Cytoadhesins, integrins, and platelets. Thromb Haemost 1988; 59: 1-6.
- 11 Parise LV. The structure and function of platelet integrins. Curr Opin Cell Biol 1989; 1: 947-52.
- 12 Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992; 69: 11-25.
- 13 Ginsberg MK, Xiaoping D, O’Toole TE, Loftus JC, Plow EF. Platelet integrins. Thromb Haemost 1993; 70: 87-93.
- 14 Shattil SJ, Brass LF. Induction of the fibrinogen receptor on human platelets by intracellular mediators. J Biol Chem 1987; 262: 992-1000.
- 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.
- 16 Shattil SJ. Regulation of platelet anchorage and signaling by integrin αIIbβ3 . Thromb Haemost 1993; 70: 224-8.
- 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.
- 18 Plow EF, Ginsberg MH. Cellular adhesion: GP IIb-IIIa as a prototypic adhesion receptor. Prog Hemost Thromb 1989; 9: 117-56.
- 19 Furman MI, Gardner TM, Goldschmidt-Clermont PJ. Mechanisms of cyto-skeletal reorganization during platelet activation. Thromb Haemost 1993; 70: 229-32.
- 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.
- 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.
- 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.
- 23 Fox JE, Phillips DR. Polymerization and organization of actin filaments within platelets. Semin Hematol 1983; 20: 243-60.
- 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.
- 25 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
- 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.
- 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.
- 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.
- 29 Fitzgerald LA, Leung B, Phillips DR. A method for purifying the platelet membrane glycoprotein IIb-IIIa complex. Anal Biochem 1985; 151: 169-77.
- 30 Stanton HC, Mersmann HJ. eds. Swine in cardiovascular research. Boca Raton, FL: CRC Press Inc.; 1986
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 37 Rucinski B, Niewiarowski S. Isolation and characterization of porcine platelets. Methods Enzymol 1989; 169: 22-6.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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.