GPIb is disulfide-linked to GPIbα to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbβ is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbβ subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbβ deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbβ, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbβ in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbβ.
Keywords
GPIb/V/IX complex -
von Willebrand factor -
adhesion
References
1
Ruggeri ZM.
Old concepts and new developments in the study of platelet aggregation. J Clin Invest 2000; 105: 699-701.
2
Kulkarni S,
Dopheide S,
Yap C,
Ravanat C,
Freund M,
Mangin P,
Heel K,
Street A,
Harper I,
Lanza F,
Jackson S.
A revised model of platelet aggregation. J Clin Invest 2000; 105: 783-91.
4
Savage B,
Saldivar E,
Ruggeri ZM.
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289-97.
5
Girma JP,
Takahashi Y,
Yoshioka A,
Diaz J,
Meyer D.
Ristocetin and botrocetin involve two distinct domains of von Willebrand factor for binding to platelet membrane GPIb. Thromb Haemost 1990; 64: 326-32.
7
Vicente V,
Houghten R,
Ruggeri ZM.
Identification of a site in the chain of platelet GPIb that participates to von Willebrand factor binding. J Biol Chem 1990; 265: 274-80.
8
De Marco L,
Mazzucato M,
Masotti A,
Ruggeri ZM.
Localization and characterization of an α thrombin-binding site on platelet GPIbα. J Biol Chem 1994; 269: 6478-84.
9
Andrews R,
Fox JEB.
Identification of a region in the cytoplasmic domain of the platelet GPIb/IX complex that binds to purified actin-binding protein. J Biol Chem 1992; 267: 18605-11.
10
Andrews R,
Harris S,
McNally T,
Berndt MC.
Binding of purified 14-3-3ζ signalling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane GPIb-IX-V complex. Biochemistry 1998; 37: 638-47.
11
Calverley D,
Kavanagh T,
Roth G.
Human signalling protein 14-3-3 interacts with platelet glycoprotein Ib subunits Ibα and Ibβ. Blood 1998; 91: 1295-303.
12
Cunningham J,
Meyer S,
Fox JEB.
The cytoplasmic domain of the α-subunit of glycoprotein (GP) Ib mediates attachment of the entire GPIb-IX complex to the cyto-skeleton and regulates von Willebrand factor induced changes in cell morphology. J Biol Chem 1996; 271: 11581-7.
14
Lopez J,
Leung B,
Reynolds C,
Li C,
Fox JEB.
Efficient plasma membrane expression of a functional platelet GPIb-IX complex requires the presence of its three subunits. J Biol Chem 1992; 267: 12851-9.
15
Lopez J,
Weisman S,
Sanan D,
Sih T,
Chambers M,
Li C.
GPIbβ is the critical subunit linking GPIbα and GPIX in the GPIb-IX complex. J Biol Chem 1994; 269: 23716-21.
17
Wardell M,
Reynolds C,
Berndt M,
Wallace R,
Fox JEB.
Platelet GPIbβ is phosphorylated on serine 166 by cyclic AMP-dependent kinase. J Biol Chem 1989; 264: 15656-61.
18
Sachs U,
Kiefel V,
Böhringer M,
Afshar-Khargan V,
Kroll H,
Santoso S.
Single amino acid substitution in human GPIbβ is responsible for the formation of the platelet-specific alloantigen Iya. Blood 2000; 95: 1849-55.
19
Toti F,
Gachet C,
Ohlmann P,
Stierle A,
Grunebaum L,
Wiesel ML,
Cazenave JP.
Electrophoretic studies on molecular defects of von Willebrand factor and platelet GPIIb-IIIa with antibodies produced in egg yolk from laying hens. Haemostasis 1992; 22: 32-40.
20
Lanza F,
Beretz A,
Stierle A,
Hanau D,
Kubina M,
Cazenave JP.
Adrenaline activates human platelets but is not an aggregating agent. Am J Physiol 1988; 225: H1276-88.
21
Ravanat C,
Freund M,
Mangin P,
Azorsa D,
Schwartz C,
Moog S,
Schuhler S,
Dambach J,
Cazenave JP,
Lanza F.
GPV is a marker of in vivo platelet activation-study in a rat thrombosis model. Thromb Haemost 2000; 83: 327-33.
22
Ulsemer P,
Lanza F,
Baas MJ,
Schwartz A,
Ravanat C,
Briquel ME,
Cranmer S,
Jackson S,
Cazenave JP,
de la Salle C.
Role of the leucine-rich domain of platelet GPIbα in correct post-translational processing: the Nancy I Bernard-Soulier mutation expressed on CHO cells. Thromb Haemost 2000; 84: 104-11.
23
Yuan Y,
Kulkarni S,
Ulsemer P,
Cranmer S,
Yap C,
Nesbitt W,
Harper I,
Mistry N,
Dopheide S,
Hughan S,
Williamson D,
de la Salle C,
Salem H,
Lanza F,
Jackson S.
The von Willebrand factor-GPIb/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and GPIb/V/IX-transfected cells. J Biol Chem 1999; 274: 36241-51.
24
Cranmer S,
Ulsemer P,
Cooke B,
Salem H,
de la Salle C,
Lanza F,
Jackson S.
GPIb-IX-transfected cells roll on a von Willebrand factor matrix under flow. J Biol Chem 1999; 274: 6097-106.
25
Coller B,
Peerschke E,
Scudder L,
Sullivan C.
Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110.
29
Yagi M,
Edelhoff S,
Disteche C,
Roth G.
Structural characterization and chromosomal location of the gene encoding human platelet GPIbβ. J Biol Chem 1994; 269: 17424-7.
30
Ware J,
Russell S,
Ruggeri ZM.
Cloning of the murine platelet GPIbα gene highlighting species-specific platelet adhesion. Blood Cells Mol Dis 1997; 23: 292-301.
31
Toda S,
Kajii Y,
Sato M,
Nishikawa T.
Reciprocal expression of infant- and adult-preferring transcripts of CDCrel-1 septin gene in the rat neocortex. Biochem Biophys Res Commun 2000; 273: 723-8.
34
Ludlow L,
Schick B,
Budarf M,
Driscoll D,
Zackai E,
Cohen A,
Konkle B.
Identification of a mutation in a GATA binding site of the platelet GPIbβ promotor resulting in the Bernard-Soulier syndrome. J Biol Chem 1996; 36: 22076-80.
35
Kenny D,
Morateck P,
Gill J,
Montgomery R.
The critical interaction of GPIbβ with GPIX-A genetic cause of Bernard-Soulier syndrome. Blood 1999; 93: 2968-75.
36
Strassel C,
Lanza F,
De la Salle C,
Baas MJ,
Cazenave JP,
Alessi MC,
Juhan-Vague I,
Pasquet JM,
Nurden P,
Nurden A.
A novel missense mutation in GPIbβ prevents the normal maturation and surface expression of the GPIb-IX-V complex in a patient with the Bernard-Soulier syndrome. Blood 2000; 96: 3525.
37
Moran N,
Morateck P,
Deering A,
Ryan M,
Montgomery R,
Fitzgerald D,
Kenny D.
Surface expression of GPIbα is dependent on GPIbβ: evidence from a novel mutation causing Bernard-Soulier syndrome. Blood 2000; 96: 532-9.
39
Kunishima S,
Lopez J,
Kobayashi S,
Imai N,
Kamiya T,
Saito H,
Naoe T.
Missense mutations of the GPIbβ gene impairing the GPIb α/β disulfide linkage in a family with giant platelet disorder. Blood 1997; 89: 2404-12.
41
Wicki A,
Clemetson K.
Structure and function of platelet membrane GPIb and GPV. Effects of leukocyte elastase and other proteases on platelets response to von Willebrand factor and thrombin. Eur J Biochem 1985; 153: 1-11.
42
Nieswandt B,
Bergmeier W,
Rackebrandt K,
Gessner JE,
Zirngibl H.
Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice. Blood 2000; 96: 2520-7.
43
Ravanat C,
Freund M,
Moog S,
Mangin P,
Azorsa D,
Schwartz C,
Lanza F,
Cazenave JP.
Differential effects of monoclonal antibodies to rat GPIIb/IIIa, GPIbα or GPV on platelet function and in vivo survival [abstract]. Thromb Haemost supplement. 1999 1660.
