Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex

Perrault, Christelle; Mangin, P; Santer, Martine; Baas, Marie-Jeanne; Moog, Sylvie; Cranmer, Susan L.; Pikovski, Inna; Williamson, David M.; Jackson, Shaun P.; Cazenave, Jean‐Pierre; Lanza, François

doi:10.1182/blood-2002-06-1847

Cited by 37 publications

(45 citation statements)

References 39 publications

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“…Whole blood (5 l) drawn from the mouse tail was diluted 1 to 10 in Tyrode's buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO 3 , 0.3 mM NaH 2 PO 4 , 1 mM MgCl 2 , 2 mM CaCl 2 , 5.5 mM glucose, 5 mM HEPES, pH 7.3) containing 0.35% human serum albumin and hirudin (100 U/ml) and incubated with an Alexa-647-conjugated rat monoclonal antibody against mouse glycoprotein Ib␤ (2 g/ml) (Perrault et al, 2003) and Alexa-488-conjugated fibrinogen (20 g/ml) for 10 min at 37°C, in the presence of either PAR-4 peptide (1 mM) or vehicle (plain Tyrode's buffer). Samples were then fixed for 20 min in plain Tyrode's buffer containing 2% paraformaldehyde.…”

Section: Measurement Of the Binding Of Alexa-488-labeled Fibrinogen Tmentioning

confidence: 99%

The Antithrombotic Activity of EP224283, a Neutralizable Dual Factor Xa Inhibitor/Glycoprotein IIbIIIa Antagonist, Exceeds That of the Coadministered Parent Compounds

Hechler¹,

Freund²,

Alame³

et al. 2011

J Pharmacol Exp Ther

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EP224283 combines in a single molecule idraparinux and tirofiban, which allows obtaining a predictable and sustained antiplatelet effect through the transfer of the pharmacokinetics properties of idraparinux to the anti-␣IIb␤3 antagonist. The activity can be instantaneously neutralized by injection of avidin, a specific antidote. We have tested the effects of this new profile anticoagulant in various thrombosis models. The antithrombotic effect of EP224283 was compared with those of the parent compounds used alone or in association at doses achieving low to moderate inhibition of platelet aggregation ex vivo. In a model of systemic thromboembolism independent of thrombin generation, tirofiban and EP224283 had similar effects at equimolar doses. On the other hand, EP224283 was more potent than tirofiban or idraparinux under thrombin-dependent conditions. In a ferric chloride-induced thrombosis model, EP224283 was more potent than either parent compound or their combination. Similar results were obtained after atherosclerotic plaque rupture in ApoE(Ϫ/Ϫ) mice. Thus, the dual action of EP224283 exceeds that of the parent compounds used in combination. A possible explanation is that EP224283 could concentrate antithrombin inside the thrombus by binding to ␣IIb␤3 through the tirofiban moiety, as shown by immunolabeling of the occluded vessel. No prolongation of the bleeding time was observed at doses achieving strong antithrombotic effects, suggesting that low to moderate ␣IIb␤3 inhibition combined with factor Xa inhibition minimizes the bleeding risk. The favorable antithrombotic profile of EP224283 together with its possible neutralization by avidin makes it an interesting drug candidate for the treatment and prevention of acute ischemic events.

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Section: Measurement Of the Binding Of Alexa-488-labeled Fibrinogen Tmentioning

confidence: 99%

The Antithrombotic Activity of EP224283, a Neutralizable Dual Factor Xa Inhibitor/Glycoprotein IIbIIIa Antagonist, Exceeds That of the Coadministered Parent Compounds

Hechler¹,

Freund²,

Alame³

et al. 2011

J Pharmacol Exp Ther

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“…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.…”

mentioning

confidence: 99%

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

Maurer

Tang

Schaff

et al. 2013

ATVB

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Objective-The glycoprotein (GP) Ib-V-IX complex regulates the adhesion, activation, and procoagulant activity of platelets.We previously reported that RAM.1, a rat monoclonal antibody directed against the extracellular domain of mouse GPIbβ, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions. Here, we further evaluated the functional importance of GPIbβ by studying the impact of RAM.1 on GPIb-mediated platelet responses and in vitro and in vivo thrombus formation. Approach and Results-We show that RAM.1 dramatically reduced GPIb-mediated filopodia extension of chinese hamster ovary GPIb-IX cells after adhesion to von Willebrand factor. RAM.1 also reduced filopodia extension and GPIb-mediated Ca 2+ signaling after adhesion of mouse platelets to von Willebrand factor. RAM.1 inhibited thrombin generation in platelet-rich plasma without impairing phosphatidylserine exposure. In addition, RAM.1 reduced thrombus formation after perfusion of mouse whole blood over collagen in a shear-dependent manner. This effect was confirmed in vivo, because injection of F(ab)′2 fragments of RAM.1 diminished thrombus formation induced by laser beam injury of mesenteric arterioles and forceps injury of the abdominal aorta. In contrast, RAM.1 F(ab)′2 did not prolong the tailbleeding time or increase the volume of blood lost. Conclusions-These

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“…[3][4][5][6][7] How this receptor complex mediates signaling in both directions is not clear, but recent work has suggested the likely interaction between the Ib␣ and Ib␤ subunits as a potential key element of the mechanism. [8][9][10] Thus, understanding the interaction between GP Ib␣ and GP Ib␤, and in general the organizing principle of the GP Ib-IX-V complex, will help to unveil the mechanism underlying transmembrane signaling mediated by the complex.It is widely accepted that the GP Ib-IX-V complex contains 7 subunits composed of 4 different polypeptides: Ib␣, Ib␤, IX, and V with a respective stoichiometry of 2:2:2:1. [11][12][13][14][15] Each subunit is a type I transmembrane protein.…”

mentioning

confidence: 99%

“…[35][36][37][38][39] Both interactions are important to the function of the GP Ib-IX-V complex. [3][4][5]8,9,40 14-3-3 is a dimer, and disrupting 14-3-3 dimerization decreases its affinity for the GP Ib-IX complex but not the isolated peptide containing the Ib␣ cytoplasmic domain. 41 On the basis of the ␣␤ heterodimeric model for GP Ib, it was thought that 2 monomers in the 14-3-3 dimer interact with the Ib␣ and Ib␤ cytoplasmic domains, respectively, with the implicit assumption that one 14-3-3 dimer interacts with one GP Ib complex.…”

mentioning

confidence: 99%

Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

Luo

Afshar-Kharghan

et al. 2006

Blood

111

129

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IntroductionPlatelet glycoprotein (GP) Ib-IX-V complex binds to von Willebrand factor (VWF) deposited at the injured blood vessel wall, mediating initial platelet rolling and adhesion to the injury site. Ligation of VWF to the GP Ib␣ subunit in the complex also transmits a signal to the platelet that leads to platelet activation and aggregation. 1,2 In addition to mediating outside-in signals, the GP Ib-IX-V complex is also capable of transmitting intracellular signals to the outside. [3][4][5][6][7] How this receptor complex mediates signaling in both directions is not clear, but recent work has suggested the likely interaction between the Ib␣ and Ib␤ subunits as a potential key element of the mechanism. [8][9][10] Thus, understanding the interaction between GP Ib␣ and GP Ib␤, and in general the organizing principle of the GP Ib-IX-V complex, will help to unveil the mechanism underlying transmembrane signaling mediated by the complex.It is widely accepted that the GP Ib-IX-V complex contains 7 subunits composed of 4 different polypeptides: Ib␣, Ib␤, IX, and V with a respective stoichiometry of 2:2:2:1. [11][12][13][14][15] Each subunit is a type I transmembrane protein. A disulfide bond links GP Ib␣ and GP Ib␤, forming a complex known as GP Ib, that in turn interacts noncovalently with GP IX and GP V to generate the Ib-IX-V complex. The Ib␣ extracellular domain contains 9 Cys residues, 7 of which reside in the N-terminal domain that contains the binding site for VWF. The N-terminal domain contains 3 disulfides, C4-C17, C209-C248, and C211-C264. 16 C65 is buried in the hydrophobic core of the N-terminal domain and is therefore unpaired. 17,18 The other 2 Cys residues in GP Ib␣, C484 and C485, are located near the transmembrane (TM) domain and are conserved across species. The Ib␣/Ib␤ ratio of 1:1 in the receptor complex predicts that only 1 of the 2 Cys residues forms a disulfide bond with the membrane-proximal C122 in GP Ib␤ (Figure 1). However, it is not clear which one is paired with C122, and, more intriguingly, what role the free thiol group of the other Cys residue plays. It seems paradoxic that 2 neighboring Cys residues are exposed to presumably similar, if not the same, oxidizing environments, yet they have entirely different fates.In the present study, we sought to assign the disulfide between GP Ib␣ and GP Ib␤. To our surprise, we found that both C484 and C485 are linked to GP Ib␤ by disulfide bonds. In other words, contrary to the currently prevailing model, 1 Ib␣ subunit is linked through disulfide bonds to 2 Ib␤ subunits. Materials and methods Vectors and antibodiesThe vector pDX 19,20 was used in the expression of GP Ib-IX complex in Chinese hamster ovary (CHO) cells. The CHO K1 cell line was obtained from ATCC (Manassas, VA). The expression vector pGEX-4T-3 was purchased from Amersham Biosciences (Piscataway, NJ). WM23, an anti-Ib␣ monoclonal antibody, was kindly provided by Dr M. Berndt. Other antibodies against individual subunits of GP Ib-IX complex, including FMC25, Gi27, SZ2, and AK2, were p...

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Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex

Abstract: Glycoprotein (GP)Ib

Cited by 37 publications

References 39 publications

The Antithrombotic Activity of EP224283, a Neutralizable Dual Factor Xa Inhibitor/Glycoprotein IIbIIIa Antagonist, Exceeds That of the Coadministered Parent Compounds

The Antithrombotic Activity of EP224283, a Neutralizable Dual Factor Xa Inhibitor/Glycoprotein IIbIIIa Antagonist, Exceeds That of the Coadministered Parent Compounds

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

Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

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