Reduced thrombin binding and aggregation in Bernard-Soulier platelets.

Jamieson, G.A.; Okumura, Tadayoshi

doi:10.1172/jci109000

Cited by 178 publications

(73 citation statements)

References 19 publications

(7 reference statements)

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“…Precipitated, GTP-bound Rap1B was separated by SDS-PAGE on 10 -20% acrylamide gradient gels, transferred to nitrocellulose, and identified by immunoblotting with a specific polyclonal antibody. Intracellular Ca 2ϩ concentration was evaluated using Fura-2-loaded platelets and calculated as described under "Experi- GPIb-IX-V on the platelet surface, and this interaction may contribute to platelet activation (33)(34)(35)(36)(37)(38). We therefore examined the role of GPIb-IX-V in thrombin-induced activation of Rap1B.…”

Section: Role Of Gpib-ix-v In the Adp-independent Activation Of Rap1bmentioning

confidence: 99%

“…This represents a high affinity receptor for thrombin, and a mounting body of evidence indicates that it may contribute to the activation of platelets (32)(33)(34)(35)(36)(37). It has been known for years that platelets from patients affected by the Bernard-Soulier syndrome, which lack GPIb-IX-V, show impaired response to thrombin (38). Moreover, platelet activation by a low concentration of thrombin is affected by antibodies against GPIb-IX-V. More recently, it has been reported that binding of thrombin to GPIb-IX-V may initiate a new pathway for platelet aggregation that does not involve PARs and is supported by polymerized fibrin (36,37).…”

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confidence: 99%

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Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

Lova

Campus

Lombardi

et al. 2004

Journal of Biological Chemistry

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Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner.

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Section: Role Of Gpib-ix-v In the Adp-independent Activation Of Rap1bmentioning

confidence: 99%

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confidence: 99%

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

Lova

Campus

Lombardi

et al. 2004

Journal of Biological Chemistry

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“…Furthermore, synthetic peptides of PAR-3 and PAR-4 that mimic the sequences of the potentially activating tethered ligands, either failed to show (PAR-3) or showed very low (PAR-4) activation effects on platelets (9,10). Therefore, attention has again turned to the GPIb-IX complex to understand, for example, why the platelets from patients with Bernard-Soulier syndrome, which lack or have dysfunctional GPIb-IX complexes on their surfaces fail to respond to low doses of thrombin (12,13).…”

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confidence: 99%

Platelet Glycoprotein Ibα Binds to Thrombin Anion-binding Exosite II Inducing Allosteric Changes in the Activity of Thrombin

Vindigni

Sadler

et al. 2001

Journal of Biological Chemistry

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The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb␣-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the aminoterminal fragment of GPIb␣ reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb␣ caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate D-Phe-Pro-Arg-pNA. The K d for the glycocalicin-thrombin interaction was 1 M at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 M at 100 mM NaCl (⌫ salt ‫؍‬ ؊4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb␣-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb␣. Both the allosteric inhibition of fibrinogen turnover caused by GPIb␣ binding to these mutants, and the K d values for their interactions with GPIb␣ were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K d values for the interactions of these thrombin mutants with GPIb␣ by 10-and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb␣. These data demonstrate that GPIb␣ binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate D-Phe-Pro-Arg-pNA.

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“…The platelets of patients with the Bernard-Soulier syndrome are deficient in GPI as well as in glycocalicin (2,8). These platelets fail to agglutinate in the presence of von Willebrand factor and ristocetin and also do not bind thrombin normally (9). The possibility has been raised that glycocalicin may act as a single receptor for ristocetin-induced and thrombin-induced platelet aggregation (10).…”

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confidence: 99%

Structural analysis of human platelet membrane glycoprotein I complex.

Nachman¹,

Kinoshita²,

Ferris³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The glycoprotein I complex, consisting of two polypeptides of Mr 210,000 and 150,000, was isolated from human platelet membranes by wheat germ lectin affinity chromatography. Glycocalicin, a soluble loosely bound membrane glycoprotein of M, 150,000 related to the glycoprotein I system, was also purified. The isolated poly eptides were radioiodinated in sodium dodecyl sulfate/! Iyac lamide gels and digested with trypsin, and the labeled Reptide digest was analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography. The two polypeptides of Mr 210,000 and 150,000 in the glycoprotein I complex had essentially identical radioactive peptide maps. Glycocalicin had a completely different t ptic peptide map. These studies shed light on the molecular relationships of some of the components of the platelet membrane glycoprotein I system. The possibility is raised that the receptorlike function of the intrinsic platelet membrane glycoproteins may be related to the polymeric subunit associations of the constituent polypeptides. The external surface glycoproteins of mammalian cells mediate important cellular reactions, including aggregation, adhesion, and surface contact interactions (1). It is therefore not surprising that platelet membrane glycoproteins have been implicated in von Willebrand factor-dependent platelet agglutination induced by ristocetin (2, 3). The glycoprotein I (GPI) complex on the surface of the human platelet may act as a von Willebrand factor receptor (4) and thus directly influence the adhesion of platelets to the subendothelium. Depending on the method of membrane solubilization and the gel system utilized for analysis, the platelet GPI complex has been found to consist of at least two distinct glycoproteins (GPIa, GPIb) (5, 6). GPIc, which is part of the GPI system, does not stain with periodic acid/Schiff reagent and is probably not a glycoprotein (6). Another constituent of the GPI complex, glycocalicin or GPIs, is easily removed from the platelet membrane by homogenization and is ordinarily detected in the soluble subcellular fractions of homogenized preparations (7). The platelets of patients with the Bernard-Soulier syndrome are deficient in GPI as well as in glycocalicin (2,8). These platelets fail to agglutinate in the presence of von Willebrand factor and ristocetin and also do not bind thrombin normally (9). The possibility has been raised that glycocalicin may act as a single receptor for ristocetin-induced and thrombin-induced platelet aggregation (10). We have previously isolated two proteins of the GPI complex from human platelet membranes by using wheat germ lectin affinity columns and demonstrated that these proteins mediate von Willebrand factor-dependent platelet agglutination induced by ristocetin (11). At present, it is not clear what the structural relationships are among the isolated separate constituents of the GPI complex. This study presents a comparison of the radioactive tryptic peptide maps of the isolated polypeptides of the platelet-GPI...

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Reduced thrombin binding and aggregation in Bernard-Soulier platelets.

Cited by 178 publications

References 19 publications

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

Platelet Glycoprotein Ibα Binds to Thrombin Anion-binding Exosite II Inducing Allosteric Changes in the Activity of Thrombin

Structural analysis of human platelet membrane glycoprotein I complex.

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