A Novel Monoclonal Antibody against the Extracellular Domain of GPIbβ Modulates vWF Mediated Platelet Adhesion

Abstract

SummaryGPIb 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.

Targeting Platelet GPIbβ Reduces Platelet Adhesion, GPIb Signaling and Thrombin Generation and Prevents Arterial Thrombosis

“…30 This study extends our previous findings by providing evidence that targeting GPIbβ with RAM.1 inhibits VWF-induced GPIb signaling both in a human and in a mouse system, as shown by decreases in filopodia extension and intracellular Ca 2+ fluxes. Interestingly, RAM.1 also impacts on TF-and collagen-induced thrombin generation.…”

“…Its binding to GPIbβ could alter the organization of the GPIb-IX complex within the plasma membrane and in turn reduce the binding properties of GPIbα, which would result in the transmission of a weaker signal. 30 Alternatively, a change in conformation could disturb interaction of GPIb-IX with intracellular partners involved in GPIbmediated signaling. This hypothesis could be supported by observation that RAM.1 promotes dephosphorylation of S166 in the intracytoplasmic domain of GPIbβ, a site implicated in 14-3-3 binding.…”

“…30,31 Although the precise mechanisms of these effects remain unclear, RAM.1 does not seem to prevent VWF binding to GPIbα through a steric hindrance notably supported by the fact that a Fab fragment of this antibody had a similar effect. 30 It has recently been reported that RAM.1 did not modify VWF binding to GPIb-IX reconstituted in phospholipid bilayer nanodiscs, further suggesting that the effect of RAM.1 may not be attributable to direct interference with the VWF-GPIbα interaction. 32 Here, we observed that RAM.1 induced a dramatic decrease in filopodia extension of hGPIb-IX CHO cells adhering to VWF, suggesting that it could also affect GPIb-mediated signals.…”

Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

“…Secondly, it is possible that phosphorylation at Ser 166 directly or indirectly induces a conformational change in the extracellular ligand-binding domain of GPIb␣. This possibility is consistent with a recent report that an anti-GPIb␤ monoclonal antibody causes inhibition of the ligand-binding function of GPIb-IX (43). Thirdly, as we showed previously that association between GPIb-IX and the membrane skeleton regulates ligandbinding function (38), it is also possible that phosphorylation of GPIb␤ induces reorganization of the GPIb-IX-associated membrane skeleton, which in turn regulates the VWF-binding function of GPIb-IX.…”

Platelet Glycoprotein Ibβ/IX Mediates Glycoprotein Ibα Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear

“…To the latter, several lines of evidence showed that when specific antibodies were used to pull down the GP Ib-IX complex, the major dimeric form is ␣/␤, instead of ␣/␣ or ␤/␤ (3,34). Because our data indicated that the ␣/␤ disulfide linkage is the major force mediating GP Ib␣ into the GEMs, an interaction being essential for platelet adhesion and platelet activation upon vWf engagement (9), an enzymatically controlled preference for ␣/␤ disulfide bond formation, as opposed to other combinations (e.g.…”