Identification of the disulphide bonds in human platelet glycocalicin

Heß, Daniel; Schaller, Johann; Rickli, Egon E.; Clemetson, Kenneth J.

doi:10.1111/j.1432-1033.1991.tb16135.x

Cited by 43 publications

(25 citation statements)

References 31 publications

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“…All the GPIb/IX/V complex subunits belong to the leucine-rich glycoprotein (LRG) family and are homologous to one another [8]. The conservation of cysteine residues in LRGs and their important roles in the folding and/or tertiary structures of the protein suggest that introduction of a Cys residue is likely to lead to abnormal conformation of GPIbb [17,18]. In fact, several mutations substituting the conserved Cys residues of GPIba (Cys209Ser) and GPIX (Cys73Tyr and Cys97Tyr) have been described in BSS [19À21].…”

Section: Discussionmentioning

confidence: 99%

Novel heterozygous missense mutation in the platelet glycoprotein Ibβ gene associated with isolated giant platelet disorder

et al. 2001

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The glycoprotein (GP) Ib/IX/V complex plays an important role in primary hemostasis, serving as the platelet receptor for von Willebrand factor (vWF). Recent studies have shown that the phenotype caused by mutations in the subunits of the GPIb/IX complex spans a wide spectrum; from the normal phenotype, to isolated giant platelet disorders (GPD), and to the full-blown bleeding disorder, the Bernard-Soulier syndrome (BSS). We characterize here a novel missense mutation of the GPIbb gene associated with isolated GPD. In the patient's platelets, the expression level of the GPIb/IX complex was moderately reduced compared with that of the GPIIb/IIIa complex, whereas the latter was expressed at higher levels than in a normal control. Immunoblot analysis showed normal electrophoretic mobility of GPIba, GPIbb, and GPIX. However, the amount of GPIbb was approximately 66% of the normal value. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIbb gene that converts Arg (CGC) to Cys (TGC) at residue 17. Transient transfection studies demonstrated that mutant GPIbb protein was not detected in transfected 293T cells. These ®ndings indicated that null expression of the abnormal GPIbb causes decreased expression of the complex and results in the GPD phenotype in the patient, and suggested that homozygosity of the mutation may lead to a

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Section: Discussionmentioning

confidence: 99%

Novel heterozygous missense mutation in the platelet glycoprotein Ibβ gene associated with isolated giant platelet disorder

et al. 2001

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“…The binding sites within the complex for both vWF and thrombin reside within the first 300 amino acids of GPIb␣ (15). GPIb␣ is also a member of a family of proteins containing leucine-rich repeat (LRR) sequences including a large number of proteins that are principally involved in mediating protein-protein interactions (16). These LRR sequences are typically 22-28 amino acids long and occur in tandem repeats that are commonly flanked by disulfide loop structures (16).…”

Section: I-labeled Thrombin To Glycocalicin Was Unaffected By the Prementioning

confidence: 99%

“…GPIb␣ is also a member of a family of proteins containing leucine-rich repeat (LRR) sequences including a large number of proteins that are principally involved in mediating protein-protein interactions (16). These LRR sequences are typically 22-28 amino acids long and occur in tandem repeats that are commonly flanked by disulfide loop structures (16). The interaction between vWF and GPIb␣ is mediated by the A1 domain of vWF and the NH 2 -terminal domain of the GPIb␣ chain (17)(18)(19)(20)(21).…”

Section: I-labeled Thrombin To Glycocalicin Was Unaffected By the Prementioning

confidence: 99%

Factor XI Interacts with the Leucine-rich Repeats of Glycoprotein Ibα on the Activated Platelet

Baglia¹,

Shrimpton

Emsley

et al. 2004

Journal of Biological Chemistry

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Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (K d app of ϳ10 nM; B max of ϳ1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn 2؉ or prothrombin and Ca I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ib␣ were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ib␤ and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ib␣ at sites comprising the leucine-rich repeat sequences within the NH 2 -terminal globular domain that are separate and distinct from the thrombin-binding site.

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“…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.…”

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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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Identification of the disulphide bonds in human platelet glycocalicin

Cited by 43 publications

References 31 publications

Novel heterozygous missense mutation in the platelet glycoprotein Ibβ gene associated with isolated giant platelet disorder

Novel heterozygous missense mutation in the platelet glycoprotein Ibβ gene associated with isolated giant platelet disorder

Factor XI Interacts with the Leucine-rich Repeats of Glycoprotein Ibα on the Activated Platelet

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

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