Download PDF
Thromb Haemost 1986; 55(02): 240-245
DOI: 10.1055/s-0038-1661529
Original Article
Schattauer GmbH Stuttgart

Glycoproteins IIb and IIIa and Platelet Thrombospondin in a Liposome Model of Platelet Aggregation

M E Rybak
The Department of Medicine, University of Massachusetts Medical Center, Worcester, MA, USA
› Author Affiliations
Further Information

Publication History

Received 19 September 1985

Accepted 12 February 1986

Publication Date:
18 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

Platelet membrane glycoproteins IIb and IIIa and platelet thrombospondin were incorporated onto phosphatidylcholine liposomes, by freeze thawing and sonication. Protein orientation on the liposomes was confirmed by susceptibility to neuraminidase cleavage and binding to lentil lectin-Sepharose (GPIIb-IIIa liposomes) and to heparin-Sepharose (thrombospondin liposomes). Glycoproteins Ilb-IIIa bound 125I-fibrinogen with Kd of 7.5 × 10™7M. Binding was reversible and calcium-dependent. Ilb-IIIa liposomes underwent fibrinogen-dependent aggregation in the presence of 10 mM CaCl2. Maximal aggregate formation was observed with a combination of IIb-IIIa liposomes and thrombospondin liposomes. This aggregation was partially inhibited by preincubation with monoclonal antibodies to the IIb-IIIa complex. Addition of EDTA caused complete reversal of aggregates. Thrombospondin liposomes also underwent fibrinogen and calcium dependent aggregation, however, this aggregation was less than that observed with the GPIIb-IIIa liposomes. Maximal aggregate formation was observed with a mixture of IIb-IIIa liposomes and thrombospondin liposomes. These studies demonstrate that GPIIb-IIIa and thrombospondin can be incorporated into phospholipid vesicles with preservation of function. Direct evidence is provided to demonstrate that glycoprotein lib and Ilia and fibrinogen are sufficient for platelet aggregation and to demonstrate that thrombospondin may also contribute to platelet aggregation.

Key words

Platelet - Thrombospondin - Glycoproteins
 

  • References

  • 1 Marguérie GA, Edgington TS, Plow EF. Human platelet possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1980; 255: 155
    PubMedGoogle Scholar
  • 2 Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 1979; 64: 1393
    CrossrefPubMedGoogle Scholar
  • 3 Peerschke EI, Zucker MB, Grant RA, Egan JJ, Johnson MM. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 1980; 55: 841
    PubMedGoogle Scholar
  • 4 Fujimura K, Phillips DR. Calcium cation regulation of glycoprotein IIb-IIIa complex formation in platelet plasma membranes. J Biol Chem 1983; 258: 10247
    PubMedGoogle Scholar
  • 5 Jennings LK, Phillips DR. Purification of glycoproteins Ha and IIIb from human platelet plasma membranes and characterization of a calcium dependent glycoprotein IIb-IIIa complex. J Biol Chem 1982; 257: 10458
    PubMedGoogle Scholar
  • 6 Phillips DR, Agin PP. Platelet membrane defects in Glanzmann’s thrombasthenia. J Clin Invest 1977; 60: 535
    CrossrefPubMedGoogle Scholar
  • 7 Nurden AT, Caen JP. The different glycoprotein abnormalities in thrombasthenic and Bernard-Soulier platelets. Sem in Hematol 1974; 28: 253
    PubMedGoogle Scholar
  • 8 Mustard JF, Kinlough-Rathbone RL, Packham MA, Perry DW, Harfenist EJ, Pai KRM. Comparison of fibrinogen associated with normal and thrombasthenic platelets on exposure to ADP or chymo-trypsin. Blood 1979; 54: 987
    PubMedGoogle Scholar
  • 9 Coller BS. Interaction of normal, thrombasthenic and Bernard-Soulier platelets withimmobilized fibrinogen. Defective platelet fibrinogen interaction in thrombasthenia. Blood 1980; 55: 169
    PubMedGoogle Scholar
  • 10 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 platelet function. Blood 1978; 51: 1065
    PubMedGoogle Scholar
  • 11 Coller BS, Peerschke EI, Scudder LE, Sullivan CA. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins in IIb and/or IIIa. J Clin Invest 1983; 72: 325
    CrossrefPubMedGoogle Scholar
  • 12 Brass LF, Shattel SJ, Bennett JS. Formation of the platelet membrane glycoprotein IIb-IIIa complex. Clin Res 1984; 32: 494A
    PubMedGoogle Scholar
  • 13 Gogstad GO, Brosstad F, Krutnes MB, Hagen I, Solum NO. Fibrinogen binding properties of the human platelet glycoprotein Ilb-Ilia complex: A study using crossed radioimmunoelectrophoresis. Blood 1982; 60: 663
    PubMedGoogle Scholar
  • 14 Bennett JS, Vilaire G, Cines DB. Identification of the fibrinogen receptor on human platelets by affinitylabeling. J Biol Chem 1982; 257: 8949
    PubMedGoogle Scholar
  • 15 Hourdille P, Hasitz M, Belloc F, Nurden AT. Immunocytochemical study of the binding of fibrinogen and thrombospondin to ADP and thrombin-stimulated human platelets. Blood 1985; 65: 912-920
    PubMedGoogle Scholar
  • 16 Levy-Toledano S, Caen JP, Breton-Gorius J. Gray platelet syndrome: alpha granule deficiency. Its influence on platelet function. J Lab Clin Med 1981; 98: 831
    PubMedGoogle Scholar
  • 17 Gerrard JM, Phillips DR, Rao GH, Plow EF, Walz DA, Ross RI, Harber LA, White JG. Biochemical studies of two patients with the gray platelet syndrome. Selective deficiency of platelet alpha granules. J Clin Invest 1980; 66: 102
    CrossrefPubMedGoogle Scholar
  • 18 Baenziger NL, Brodie GN, Majerus PW. A thrombin-sensitive protein of human platelet membranes. Proc Natl Acad Sci USA 1971; 68: 240
    CrossrefPubMedGoogle Scholar
  • 19 Lawler JW, Slayter HS, Coligan JE. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J Biol Chem 1981; 256: 7495
    PubMedGoogle Scholar
  • 20 Nurden AT, Kunicki TJ, Dupuis P, Soria C, Caen JP. Specific protein and glycoprotein deficiencies in platelets isolated from two patients with the gray platelet syndrome. Blood 1982; 59: 709
    PubMedGoogle Scholar
  • 21 Phillips DR, Jennings LK, Prasanna HR. Ca+2 mediated association of glycoprotein G (thrombin sensitive protein thrombospondin) with human platelets. J Biol Chem 1980; 255: 11629
    PubMedGoogle Scholar
  • 22 Agam G, Shohat O, Livne A. Thrombospondin plays a role in platelet-platelet recognition during release related aggregation. J Soc Exp Biol Med 1982; 177: 482
    PubMedGoogle Scholar
  • 23 Leung LK. Role of thrombospondin in platelet aggregation. J Clin Invest 1985; 74: 1764-1772
    PubMedGoogle Scholar
  • 24 DiPaola M, Maxfield FR. Conformational changes in receptors for epidermal growth factor and Asialo-glycoproteins induced by mildly acidic pH found in endocytic vesicles. J Biol Chem 1984; 258: 9163
    PubMedGoogle Scholar
  • 25 Wheeler TJ, Hinkle PC. Kinetic properties of the reconstituted glucose transporter from human erythrocytes. J Biol Chem 1981; 256: 8907
    PubMedGoogle Scholar
  • 26 Kassahara M, Hinkle PC. Reconstitution and purification of the D-glucose transporter from human erythrocytes. J Biol Chem 1977; 252: 7384
    PubMedGoogle Scholar
  • 27 Leung LLK, Kinoshita T, Nachman RL. Isolation, purification and partial characterization of platelet membrane glycoproteins IIb-IIIa. J Biol Chem 1981; 256: 1954
    PubMedGoogle Scholar
  • 28 Clemetson KJ, Pfueller SL, Liischer EF, Jenkins CS P. Isolation of the membrane glycoproteins of blood platelets by lectin affinity chromatography. Biochim Biophys Acta 1977; 464: 493
    CrossrefPubMedGoogle Scholar
  • 29 Sixma JJ, Schiphorst ME. Identification of ectoproteins of human platelets. Combination of radioactive labelling and two dimensional electrophoresis. Biochem Biophys Acta 1980; 603: 70
    CrossrefPubMedGoogle Scholar
  • 30 Margossian SS, Lawler JW, Slater HS. Physical characterization of platelet thrombospondin. J Biol Chem 1981; 256: 7495
    PubMedGoogle Scholar
  • 31 Dixit PI, Grant GA, Santoro SA, Frazier WA. Isolation and characterization of heparin binding domain from the amino terminal of platelet thrombospondin. J Biol Chem 1984; 259: 10100
    PubMedGoogle Scholar
  • 32 Doolittle RF, Cassman KG, Cottrel BA, Freizner SJ, Hucko JT, Takagi T. Amino acid sequence studies of the chain of human fibrinogen. Characterization of Cyanogen bromide fragments. Biochem 1977; 16: 1703
    CrossrefPubMedGoogle Scholar
  • 33 Parise LV, Phillips DR. Platelet membrane glycoprotein IIb-IIIa complex, incorporated into phospholipid vesicles: preparation and morphology. J Biol Chem 1985; 260: 1750
    PubMedGoogle Scholar
  • 35 Phillips DR, Baughan AK. Fibrinogen binding to human platelet plasma membranes. J Biol Chem 1983; 258: 10240
    PubMedGoogle Scholar
  • 35 Baldassare JJ, Kahn RA, Knipp MA, Newman PJ. Reconstitution of platelet proteins into phospholipid vesicles. J Clin Invest 1985; 75: 35
    CrossrefPubMedGoogle Scholar
  • 36 Parise LV, Phillips DR. Reconstitution of the purified platelet fibrinogen receptor. J of Biol Chem 1985; 260: 10698-10707
    PubMedGoogle Scholar
  • 37 Ruggeri ZM, DeMarco L, Gatti L, Bader R, Montgomery RR. Platelets have more than one binding site for von Willebrand factor. J Clin Invest 1983; 72: 1
    CrossrefPubMedGoogle Scholar
  • 38 Dixit VM, Haverstick DM, O’Rourke KM, Hennessy SW, Grant GA, Santoro SA, Frazier WA. A monoclonal antibody against human thrombospondin inhibits platelet aggregation. Proc Natl Acad Sci 1985; 82: 3472-3476
    CrossrefPubMedGoogle Scholar