Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ibβ prevents the normal maturation and surface exposure of GPIb-IX complexes

Consuelo González-Manchón; Nora Butta; Gema Iruín; Sonia Alonso; Matilde S. Ayuso; Roberto Parrilla

doi:10.1160/TH03-01-0026

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 90(03): 456-464
DOI: 10.1160/TH03-01-0026

Platelets and Blood Cells

Schattauer GmbH

Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ibβ prevents the normal maturation and surface exposure of GPIb-IX complexes

Authors

Consuelo González-Manchón

²Department of Pathophysiology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain
Nora Butta

²Department of Pathophysiology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain
Gema Iruín

¹Department of Hematology, Hospital de Cruces, Baracaldo, Bilbao, Spain
Sonia Alonso

²Department of Pathophysiology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain
Matilde S. Ayuso

²Department of Pathophysiology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain
Roberto Parrilla

²Department of Pathophysiology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain

Abstract

Summary

This work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ibα and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIbα, GPIbβ, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIbβ, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIbα and GPIX. Transient transfection of the mutant GPIbβ subunit failed to render surface expression of GPIbα and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIbβ may associate with GPIX and GPIbα, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5-Cys7 disulfide loop in GPIbβ is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIbβ mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.

Keywords

Gene mutations - inherited/acquired platelet disorders - platelet physiology - protein trafficking - protein structure/folding

Full Text

References

References
1 Andrews RK, López JA, Berndt MC. Molecular mechanisms of platelet adhesion and activation. Int J Biochem Cell Biol 1997; 29: 91-105.
2 Kroll MH, Hellums JD, McIntire LV, Schafer AI, Moake JL. Platelets and shear stress. Blood 1996; 88: 1525-41.
3 López JA. The platelet glycoprotein Ib-IX complex. Blood Coagul Fibrinolysis 1994; 5: 97-119.
4 Nurden AT, Caen JP. Specific roles for platelet surface glycoprotein in platelet function. Nature 1975; 255: 720-2.
5 López JA, Andrews RK, Afshar-Kharghan V, Berndt MC. Bernard-Soulier syndrome. Blood 1998; 91: 4397-418.
6 Kahn ML, Diacovo TG, Bainton DF, Lanza F, Trejo J, Coughlin SR. Glycoprotein V-deficient platelets have undiminished thrombin responsiveness and do not exhibit a Bernard-Soulier phenotype. Blood 1999; 94: 4112-21.
7 López 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: 12851-9.
8 Ulsemer P, Strassel C, Baas M-J. et al. Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX. Biochem J 2001; 358: 295-303.
9 Dong J, Gao S, López JA. Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex. J Biol Chem 1998; 273: 31449-54.
10 Wu G, Meloni FJ, Shapiro SS. Platelet glyco-protein (Gp) IX associates with GPIb alpha in the platelet membrane GPIb complex. Blood 1996; 87: 2782-7.
11 Kenny D, Morateck PA, Gill JC, Montgomery RR. The critical interaction of glycoprotein (GP) Ibβ with GPIX. A genetic cause of Bernard-Soulier syndrome. Blood 1999; 93: 2968-75.
12 Moran N, Morateck PA, Deering A. et al. Surface expression of glycoprotein Ibα is dependent on glycoprotein Ibβ: evidence from a novel mutation causing Bernard-Soulier syndrome. Blood 2000; 96: 532-9.
13 López JA, Weisman S, Sanan DA, Sih T, Chambers M, Li CQ. Glycoprotein (GP) Ibβ is the critical subunit linking GPIbα and GPIX in the GPIb-IX complex. Analysis of partial complexes. J Biol Chem 1994; 269: 23716-21.
14 Perrault C, Moog S, Rubinstein E. et al. A novel monoclonal antibody against the extra-cellular domain of GPIbβ modulates vWF mediated platelet adhesion. Thromb Haemost 2001; 86: 1238-48.
15 Kenny D, Morateck PA, Montgomery RR. The cysteine knot of platelet glycoprotein Ibβ (GPIbβ) is critical for the interaction of GPIbβ with GPIX. Blood 2002; 99: 4428-33.
16 González-Manchón C, Larrucea S, Pastor AL. et al. Compound heterozygosity of the GPIbα gene associated with Bernard-Soulier syndrome. Thromb Haemost 2001; 86: 1385-91.
17 López JA, Li CQ, Weisman S, Chambers M. The glycoprotein Ib-IX complex-specific monoclonal antibody SZ1 binds to a conformation-sensitive epitope on glycoprotein IX: Implications for the target antigen of quinine/quinidine-dependent autoantibodies. Blood 1995; 85: 1254-8.
18 Ludlow LB, Schick BP, Budarf ML. et al. Identification of a mutation in a GATA binding site of the platelet glycoprotein Ibα promoter resulting in the Bernard-Soulier syndrome. J Biol Chem 1996; 271: 22076-80.
19 Kunishima S, Matsushita T, Ito T, Kamiya T, Saito H. Novel nonsense mutation in the platelet glycoprotein Ib beta gene associated with Bernard-Soulier syndrome. Am J Hematol 2002; 71: 279-84.
20 Kunishima S, Naoe T, Kamiya T, Saito H. Novel heterozygous missense mutation in the platelet glycoprotein Ib beta gene associated with isolated giant platelet disorder. Am J Hematol 2001; 68: 249-55.
21 Hillmann A, Nurden A, Nurden P. et al. A novel hemizygous Bernard-Soulier syndrome (BSS) mutation in the amino terminal domain of glycoprotein (GP)Ib beta. Platelet characterization and transfection studies. Thromb Haemost 2002; 88: 1026-32.
22 Strassel C, Pasquet JM, Alessi MC. et al. A novel missense mutation shows that GPIbbeta has a dual role in controlling the processing and stability of the platelet GPIb-IX adhesion receptor. Biochemistry 2003: 4452-62.
23 Kunishima S, Tomiyama Y, Honda S. et al. Homozygous Pro74->Arg mutation in the platelet glycoprotein Ibβ gene associated with Bernard-Soulier syndrome. Thromb Haemost 2000; 84: 112-7.
24 Kurokawa Y, Ishida F, Kamijo T. et al. A mis-sense mutation (Tyr88 to Cys) in the platelet membrane glycoprotein Ibβ gene affects GPIb/IX complex expression. Bernard-Soulier syndrome in the homozygous form and giant platelets in the hetrozygous form. Thromb Haemost 2001; 86: 1249-56.
25 Rivera CE, Villagra J, Riordan M, Williams S, Lindstrom KJ, Rick ME. Identification of a new mutation in platelet glycoprotein IX (GPIX) in a patient with Bernard-Soulier syndrome. Br J Haematol 2001; 112: 105-8.