Thrombin Receptors of Human Platelets: Thrombin Binding and Antithrombin Properties of Glycoprotein I

Ganguly, P.; Gould, N. L.

doi:10.1111/j.1365-2141.1979.tb03706.x

Cited by 62 publications

(26 citation statements)

References 20 publications

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“…Ganguly and Gould (11) demonstrated that GPIb isolated by lectinSepharose affinity chromatography specifically and reversibly bound 125I-labeled thrombin. Thrombin binds to immobilized and free glycocalicin, a proteolytic fragment of GPIba, and glycocalicin acts as a competitive inhibitor of thrombin binding to platelets (10,12).…”

Section: Discussionmentioning

confidence: 99%

High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

Gralnick¹,

Williams²,

McKeown³

et al. 1994

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

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The platelet thrombin binding site(s) has been a controversial issue. Vu and coworkers (4-6) have cloned a platelet thrombin receptor which is a member of the 7-transmembrane loop-receptor family. This receptor is cleaved by thrombin between residues R41 and S42, exposing a new amino-terminal peptide, which serves as a tethered ligand whose binding site resides within the first six amino acids. This tethered ligand peptide binds to an undefined site that induces receptor activation (7-9).Prior to the discovery and cloning of the seven-transmembrane loop receptor, several studies had defined platelet glycoprotein lb (GPIb) as an a-thrombin receptor. These studies described the binding of thrombin to GPIb and the inhibition of a-thrombin binding to GPIb by glycocalicin (a proteolytic fragment of GPIba) and monoclonal antibodies directed against . The decrease or absence of high-affinity a-thrombin binding to Bernard-Soulier platelets, which lack GP~ba (17), provided further evidence that GPIb was a thrombin receptor. In experiments using crosslinking of a-thrombin to human platelets, the only identifiable site of binding was GPIb (18,19).The proposed roles of GPIba in thrombin binding include acting as a high-affinity receptor for localizing a-thrombin near the cell surface or serving as a site for excess thrombin binding (20). In this scheme, GPIba acts primarily as a neutralizing site for excess thrombin generation or as an independent modulator of platelet function through an undefined mechanism. We have studied the high-affinity binding of a-thrombin to human platelets and have examined the inhibitory effects of synthetic peptides derived from portions of the sequence of the GPIba chain and from the seventransmembrane loop thrombin receptor in order to assess the site(s) of high-and moderate-affinity thrombin binding to platelets and thrombin-induced platelet aggregation.MATERIALS AND METHODS Platelet Preparation. Blood was obtained by venipuncture using a two-syringe technique. The blood from the second syringe was placed into polypropylene tubes containing 0.1 ml of sodium citrate anticoagulant, final concentration 10.9 pM. Platelet-rich plasma was prepared from whole blood after centrifugation at 750 x g for 3 min at 250C. Then 6-10 ml of platelet-rich plasma was placed on a discontinuous Larcoll (Sigma) gradient, 20%o (3 ml) and 10% (5 ml), and the platelets were separated from the plasma proteins by centrifugation at 2000 x g for 30 min. The platelets were then resuspended in Hepes buffer at pH 7.35. All platelets were used within 2 hr of being obtained.Thrombin Lalin. Human a-thrombin was prepared and radiolabeled as described (21). Two hundred fifty microliters of a-thrombin solution (1 pg/2 units of activity) was treated with 5 Ad oflactoperoxidase beads, 5 td of0.5 mM KI, 0.5 mCi (18.5 MBq) of Na125I, and 1 A4 of 0.015% H202 for 5 min at room temperature. Free 125I was separated from bound 125I on a 10-ml column of Sephadex G-25 fine equilibrated with 0.05 M Tris/0.1 M NaCl at pH 7.35. Fractions were ...

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Section: Discussionmentioning

confidence: 99%

High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

Gralnick¹,

Williams²,

McKeown³

et al. 1994

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

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“…The a-chain remnant has probably remained unidentified for so long because it could not be labeled with any surface-labeling method and only the combined use of a rabbit anti-GPIbfl IgG affinity column, non-reducedlreduced 2 D SDS-PAGE and the development of sensitive silver-staining methods allowed its detection and isolation. GPIb is known to contain a thrombinbinding site [5,40]. Cleavage ofpurified glycocalicin by human leukocyte elastase gave two polypeptides of 90 kDa and 45 kDa.…”

Section: Discussionmentioning

confidence: 99%

The glycoprotein Ib complex of human blood platelets

Wicki

Clemetson

1987

European Journal of Biochemistry

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Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1 YO sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIba, which is very rich in 0-linked oligosaccharides, sugar analysis revealed that GPIbg and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal a-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one-or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIbP by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPTba. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.Blood platelets play a crucial role in the initial event of hemostasis by adhering to exposed vascular subendothelium [l, 21. This complex process is mediated by binding of a plasma component, von Willebrand factor, first to exposed subendothelium and then to a receptor on platelets, the membrane glycoprotein Ib [3, 41. Glycoprotein Ib is also involved in activation of platelets by thrombin [5-71 and in binding of quininelquinidine-dependent antibodies [8 -101. Glycoprotein Ib has an apparent molecular mass of 165 kDa and consists of two subunits, GPIba and GPIbB, with molecular masses of 135 kDa and 25 kDa, linked by a disulfide bridge [Ill. Most of the information about GPIb is derived from studies with glycocalicin, a major proteolytic fragment [12, 131. Glycocalicin can itself be split by various proteases into two pieces, a carbohydrate-rich 90-kDa fragment and a 45-kDa fragment which contains the receptor sites for thrombin and von Willebrand factor [6, 241. GPIb has all the characteristics of a typical receptor and probably functions as part of a complex containing several types of molecules.

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“…The first receptor for thrombin identified on the platelet surface was the glycoprotein (GP) 1 Ib-IX complex (3)(4)(5)(6), although the biological significance of this interaction remains unknown 20 years later. In the interim, other thrombin receptors have also been identified on the platelet surface including three members of the seven transmembrane domain receptor superfamily known as proteolytically activated receptor-1 (PAR-1), PAR-3, and PAR-4 (7-10).…”

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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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Thrombin Receptors of Human Platelets: Thrombin Binding and Antithrombin Properties of Glycoprotein I

Cited by 62 publications

References 20 publications

High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

The glycoprotein Ib complex of human blood platelets

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

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