Download PDF
Thromb Haemost 1996; 76(05): 768-773
DOI: 10.1055/s-0038-1650658
Original Article
Schattauer GmbH Stuttgart

Functional Expression of Single Chain Glycoprotein lb Alpha on the Surface of COS Cells and BHK Cells

Eefke J Petersen
The Department of Haematology, University Hospital Utrecht, The Netherlands
,
Eelke Posthumus
The Department of Haematology, University Hospital Utrecht, The Netherlands
,
Jan J Sixma
The Department of Haematology, University Hospital Utrecht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 06 January 1995

Accepted after resubmission 21 June 1996

Publication Date:
11 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

The GpIb-IX complex constitutes the major receptor for von Wille-brand factor (vWF) on the surface of blood platelets. The vWF-binding site has been mapped to one of the three constituent chains, Gplba. Surface expression of Gplba depends on the. correct intracellular assembly with the Gplb(3 and GpIX chains. We have now grafted a portion of the extracellular domain of Gplba containing the vWF binding site onto the transmembrane/intracellular domain of the single chain surface molecule ICAM-1. Transient transfection of this chimeric protein in COS cells resulted in surface expression as assessed by immunostain-ing of live cells. Similar results were obtained after stable transfection into BHK cells. Purified vWF bound to the surface of transfected cells in the presence of ristocetin and botrocetin with a Kd of 52 ng/ml, comparable to the Kd for fixed platelets (65.5 ng/ml). This study indicates that functional expression of the vWF-binding domain of GpIbα on the surface of mammalian cells can be obtained in the absence of GpIbβ and GpIX. Furthermore, this model system simplifies existing methods for the assessment of the functional consequences of mutations in Gplba as found in pseudo-von Willebrand disease and Bemard-Soulier syndrome.

 

  • References

  • 1 Weiss HJ, Turitto VT, Baumgartner HR. Effects of shear rate on platelet interaction with subendothelium in citrated and native blood. Shear-dependent decrease of adhesion in von Willebrand’s disease and Bernard-Soulier syndrome. J Lab Clin Med 1978; 92: 750-764
    PubMedGoogle Scholar
  • 2 Weiss HJ, Tschopp TB, Baumgartner HR, Sussman IL, Johnson MM, Egan JJ. Decreased adhesion of giant (Bernard-Soulier) platelets to subendothelium. Further implications on the role of the von Willebrand factor in hemostasis. Am J Med 1974; 57: 920-925
    CrossrefPubMedGoogle Scholar
  • 3 Sixma JJ, Sakariassen KS, Stel HV, Houdijk WPM, In de Maur DW, Hamer RJ, de Groot PG, van Mourik JA. Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and collagen. J Clin Invest 1984; 74: 736-744
    PubMedGoogle Scholar
  • 4 Girma JP, Kalafatis M, Pietu G, Lavergne JM, Chopek MW, Edgington TS, Meyer D. Mapping of distinct von Willebrand factor domains interacting with platelet Gplb and GpIIb/IIIa and with collagen using monoclonal antibodies. Blood 1986; 67: 1356-1366
    PubMedGoogle Scholar
  • 5 Berndt MC, Gregory C, Kabral A, Zola A, Fournier D, Castaldi A. Purification and preliminary characterization of the glycoprotein lb complex in the platelet membrane. Eur J Biochem 1985; 152: 637-649
    PubMedGoogle Scholar
  • 6 Wicki AN, Clemetson KJ. The glycoprotein lb complex of human blood platelets. Eur J Biochem 1987; 163: 43-50
    CrossrefPubMedGoogle Scholar
  • 7 Lopez JA, Chung DW, Fujikawa K, Hagen FS, Papayannopoulou T, Roth GJ. Cloning of the alpha chain of human platelet glycoprotein lb: a transmembrane protein with homology to leucine-rich α2-glycoprotein. Proc Natl Acad Sci USA 1987; 84: 5615-5619
    CrossrefPubMedGoogle Scholar
  • 8 Lopez JA, Chung DW, Fujikawa K, Hagen FS, Davie EW, Roth GJ. The α and β chains of glycoprotein lb are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc Natl Acad Sci USA 1988; 85: 2135-2139
    CrossrefPubMedGoogle Scholar
  • 9 Hickey MJ, Williams SA, Roth GJ. Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures. Proc Natl Acad Sci USA 1989; 86: 6773-6777
    CrossrefPubMedGoogle Scholar
  • 10 Hickey MJ, Hagen FS, Mayumi Y, Roth GJ. Human platelet glycoprotein V: Characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins. Proc Natl Acad Sci USA 1993; 90: 8327-8331
    CrossrefPubMedGoogle Scholar
  • 11 Roth GJ. Developing relationships: Arterial platelet adhesion, Glycoprotein lb, and Leucine-rich glycoproteins. Blood 1991; 77: 5-19
    PubMedGoogle Scholar
  • 12 Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM. The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. J Biol Chem 1986; 261: 12579-12585
    PubMedGoogle Scholar
  • 13 Vincente V, Houghten RA, Ruggeri ZM. Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand Factor binding. J Biol Chem 1990; 265: 274-280
    PubMedGoogle Scholar
  • 14 Okumura T, Jamieson GA. Platelet Glycocalicin I. Orientation of glycoproteins of the human platelet surface. J Biol Chem 1976; 251: 5944-5950
    PubMedGoogle Scholar
  • 15 Michelson AD, Loscalzo J, Melnick B, Coller BS, Handin RI. Partial characterization of a binding site for von Willebrand’s factor on glycocalicin. Blood 1986; 67: 19-24
    PubMedGoogle Scholar
  • 16 Fox JE, Lawrence P, Aggerbeck LP, Berndt MC. Structure of the glycoprotein Ib/IX complex from platelet membranes. J Biol Chem 1988; 263: 4882-4890
    PubMedGoogle Scholar
  • 17 Okumura T, Lombart C, Jamieson GA. Platelet glycocalicin II. Purification and characterization. J Biol Chem 1976; 251: 5950-5955
    PubMedGoogle Scholar
  • 18 Vincente V, Houghten RA, Ruggeri ZM. Identification of a site in the alpha chain of platelet glycoprotein lb that participates in von Willebrand factor binding. J Biol Chem 1990; 265: 274-280
    PubMedGoogle Scholar
  • 19 Katagiri Y, Hayashi Y, Yamamoto K, Tonoue K, Kosaki G, Yamazaki H. Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib. Thromb Haemost 1990; 63: 122-126
    Article in Thieme ConnectPubMedGoogle Scholar
  • 20 Petersen EJ, Cruz MA, Turci SM, Robert RI. Functional analysis of a recombinant glycoprotein lb alpha polypeptide, which inhibits von Willebrand Factor binding to the platelet GpIb/IX complex and to collagen. J Biol Chem 1992; 267: 1303-1307
    PubMedGoogle Scholar
  • 21 Petersen EJ, Handin RI. Transient expression of recombinant Glycoprotein lb alpha polypeptides in COS cells, that inhibit von Willebrand Factor binding to the platelet Glycoprotein Ib/IX complex. Thromb Haemost 1992; 68: 203
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Miller JL, Hustad KO, Kupinski JM, Lyle VA, Kunicki TJ. Increased platelet sensitivity to ristocetin is predicted by the binding characteristics of a GpIb/IX determinant. Br J Haematol 1990; 74: 313-319
    CrossrefPubMedGoogle Scholar
  • 23 Weiss HJ, Meyer D, Rabinowitz R, Girma JP, Vicic WJ, Rogers J. Pseudo-von Willebrand’s disease. An intrinsic platelet defect with aggregation by unmodified human Factor VUI/von Willebrand Factor and enhanced adsorption of its high molecular weight multimers. N Eng J Med 1982; 306: 326-333
    PubMedGoogle Scholar
  • 24 Miller JL, Cunningham D, Lyle VA, Finch CN. Mutation in the gene encoding the alpha chain of platelet glycoprotein lb in platelet type von Willebrand disease. Proc Natl Acad Sci 1991; 88: 4761-4765
    CrossrefPubMedGoogle Scholar
  • 25 Russel SD, Roth GJ. Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor. Blood 1993; 81: 1787-1791
    PubMedGoogle Scholar
  • 26 Bernard J, Soulier J-P. Sur une nouvelle variete de dystrophie thrombo-cytaire-hemorrhagipare congenitale. Semin Hop Paris 1948; 24: 3217-3223
    PubMedGoogle Scholar
  • 27 Lopez JA, Leung B, Reynolds CC, Li CQ, Fox JEB. Efficient plasma membrane expression of a functional platelet glycoprotein Ib/IX complex requires the presence of its three subunits. J Biol Chem 1992; 267: 12851-12855
    PubMedGoogle Scholar
  • 28 Klausner RD, Lippincott-Schwartz J, Bonifacino JS. The T cell antigen receptor: insights into organelle biology. Annu Rev Cell Biol 1990; 6: 304-333
    PubMedGoogle Scholar
  • 29 Alarcon B, Berkhout B, Breitmeyer J, Terhorst C. Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma-delta-epsilon core and single T cell receptor alpha or beta chains. J Biol Chem 1988; 263: 2953-2961
    PubMedGoogle Scholar
  • 30 Sancho J, Chatila T, Wong RCK, Hall G, Blumberg R, Alarcon B, Geha RS, Terhorst C. T-cell antigen receptor (TCR)-alpha/beta heterodimer formation is a prerequisite for association of CD3-zeta2 into functionally competent TCR-CD3 complexes. J Biol Chem 1989; 264: 20760-20769
    PubMedGoogle Scholar
  • 31 Wileman T, Carson GR, Concino M, Ahmed A, Terhorst C. Gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors. J Cell Biol 1990; 110: 973-986
    CrossrefPubMedGoogle Scholar
  • 32 Coller BS, Peerschke El, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: Additional evidence in support of Gplb as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110
    PubMedGoogle Scholar
  • 33 Kroll MH, Harris TS, Moake JL, Handin RI, Schafer AI. Von Willebrand factor binding to platelet Gplb initiates signals for platelet activation. J Clin Invest 1991; 88: 1568-1573
    CrossrefPubMedGoogle Scholar
  • 34 Aruffo A, Seed B. Molecular cloning of a CD28 cDNA by a high efficiency COS cell expression system. Proc Natl Acad Sci USA 1987; 84: 8573
    CrossrefPubMedGoogle Scholar
  • 35 Ware J, Russell SR, Marchese P, Murata M, Mazzucato M, De Marco M, Ruggeri ZM. Point mutation in a leucine-rich repeat of platelet Gplba resulting in the Bernard-Soulier syndrome. J Clin Invest 1992; 92: 1213-1220
    PubMedGoogle Scholar
  • 36 Lopez JA, Weisman S, Sanan DA, Sih T, Chambers M, Li CQ. Glycoprotein (GP) Ib(3 is the critical subunit linking Gplba and GpIX in the GpIb-IX complex. J Biol Chem 1994; 269: 23716-23721
    PubMedGoogle Scholar
  • 37 Meyer S, Kresbach G, Haring P, Schumpp-Vonach B, Clemetson KJ, Hadvary P, Steiner B. Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. J Biol Chem 1993; 268: 20555-20562
    PubMedGoogle Scholar
  • 38 Murata M, Russel SR, Ruggeri ZM, Ware J. Expression of the phenotypic abnormality of platelet-type von Willebrand disease in a recombinant Glycoprotein Iba fragment. J Clin Invest 1993; 91: 2133-2137
    CrossrefPubMedGoogle Scholar