Retention of glycoprotein Ib/IX receptors on external surfaces of thrombin-activated platelets in suspension

White, JG; Krumwiede,; Cocking-Johnson, Debra; Rao, G. H. R.; Escolar, G

doi:10.1182/blood.v86.9.3468.bloodjournal8693468

Cited by 19 publications

(8 citation statements)

References 14 publications

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“…Results of the present investigation have shown that GPIb receptors are homogeneously distributed on external and internal membranes of resting platelets. A similar concept was suggested by early studies, [12][13][14][15][16][17] but the difficulty in labeling GPIb on membranes in narrow channels of the OCS limited a final conclusion. The extensive system of dilated OCS channels and large membrane complexes in giant platelets from patients with the May-Hegglin anomaly 31,32 and Epstein's syndrome 33 made them suitable candidates for evaluating the distribution of GPIb on resting platelet membranes.…”

Section: Discussionmentioning

confidence: 60%

“…Some authors have found that the density of labeling for GPIb within the OCS is less than that observed on the platelet surface. 15,16 Other workers have failed to note significant differences, 12,17 whereas some using enzyme-linked immunsorbent assay techniques with monoclonal antibodies have predicted a large internal pool of GPIb that could replenish any amount of GPIb lost from the external membrane. 18 The latter authors estimated the number of internal copies of GPIb was 3 to 4 times greater than the number of GPIb molecules present on the platelet surface, figures relatively higher than those estimated for GPIIb-IIIa.…”

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confidence: 99%

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Glycoprotein Ib Is Homogeneously Distributed on External and Internal Membranes of Resting Platelets

White

Krumwiede

Escolar

1999

The American Journal of Pathology

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Recent ultrastructural studies have suggested that Glycoprotein Ib (GPIb) has a different distribution on external (surface) versus internal (open canalicular system) membranes in resting discoid platelets. The differential distribution proposed for GPIb differs from that reported for the fibrinogen receptor, GPIIb-IIIa, and could have profound physiological significance when platelets are activated by surfaces. The present study explored the distribution of GPIb on external and internal membranes of resting platelets. Immunogold cytochemical techniques were applied to ultrathin cryosections of washed platelets. Polyclonal antibodies or mixtures of monoclonal antibodies (AP1 and 6D1) were used for labeling. To avoid the technical problem posed by limited accessibility of antigens located in very narrow portions of the open canalicular system (OCS) to antibodies, the same methods were applied to patients with giant platelets syndromes. The OCS of normal resting platelets was also dilated by exposure of platelets to hypertonic conditions or to cytochalasin-B, an agent that prevents assembly of actin, and, reportedly, movement of GPIb. Morphometric analysis revealed that rates of labeling on internal versus external membranes of giant platelets does not differ significantly (0.93 +/- 0.20), provided the OCS is sufficiently dilated. Platelets exposed to cytochalasin B (1.01 +/- 0.31) or to hypertonic conditions (0.96 +/- 0.20) revealed similar ratios for immunogold particles on external and internal membranes. Results of our study indicate that membranes of the exposed surface and lining OCS channels of resting platelets are continuous, identical structures and GPIb is homogeneously distributed on external and internal membranes.

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Section: Discussionmentioning

confidence: 60%

mentioning

confidence: 99%

Glycoprotein Ib Is Homogeneously Distributed on External and Internal Membranes of Resting Platelets

White

Krumwiede

Escolar

1999

The American Journal of Pathology

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“…Immunoelectron microscopy has shown that this loss is associated with an accumulation of GPIb–IX–V within the SCCS, a membrane system that ramifies throughout the platelet [15,35]. Nevertheless, a transitional internalization of GPIb in response to stimulation is still controversial [18,25,36]. In our current work, we have used PFA‐fixed platelets to avoid artifactual GPIb movement by receptor cross‐linking, as was already suggested [37].…”

Section: Discussionmentioning

confidence: 98%

Redistribution of glycoprotein Ib within platelets in response to protease-activated receptors 1 and 4: roles of cytoskeleton and calcium

Han

Nurden

Combrié

et al. 2003

Journal of Thrombosis and Haemostasis

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Summary. Thrombin activates human platelets by hydrolyzing the protease‐activated receptors PAR‐1 and PAR‐4, exposing new N‐terminal sequences which act as tethered ligands, and binding to glycoprotein (GP) Ib, whose surface accessibility transiently decreases when platelets are stimulated by the enzyme. In an attempt to better understand this latter process, we used the peptides SFLLRNPNDKYEPF (PAR‐1‐AP or TRAP) and AYPGKF (PAR‐4‐AP) to study whether hydrolysis of both PAR receptors leads to GPIb redistribution. Both peptides induced surface clearance of GPIb with a maximum at 2 min and 5 min for PAR‐1‐AP and PAR‐4‐AP, respectively, followed by a slow return to the surface with levels normalizing between 30 and 60 min. Translocation was associated with the formation of clusters of GPIb as revealed by fluorescence microscopy. This transient redistribution of GPIb was blocked by cytochalasin D and in large part by the membrane permeable Ca2+ chelator, BAPTA. The inhibitor of phosphatidylinositol 3‐kinase and myosin light chain kinase, wortmannin, did not significantly modify internalization of GPIb, although its return to the surface was delayed for PAR‐1‐AP. PAR receptor–mediated association of GPIb to the insoluble cytoskeleton was blocked by cytochalasin D, while BAPTA alone increased and stabilized the presence of GPIb. Globally, immunoprecipitation experiments and analysis of the cytoskeleton confirmed that GPIb translocation is powered by a contractile mechanism involving Ca2+ mobilization, actin polymerization, and myosin incorporation into the cytoskeleton and that both PAR‐1 and PAR‐4 can activate this process.

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“…It came as a surprise when using platelets stimulated by I U/ml thrombin in a medium without Ca2* or in the presence of 5 mM EDTA and a polyclonal antibody to glycocalicin, Escolar et al (32) failed to detect any changes in the surface density of GP Ib either by immunogold staining and electron microscopy or by flow cytometry. White et al (33) reported likewise following the immunolabelling of cryosections of platelets stimulated with lower doses of thrombin for up to 30 min. Furthermore, when platelets were preincubated with two murine MoAbs to GP Ib, AP-1 and 6D1, prior to their activation with thrombin, the bulk of the murine antibodies remained on the surface (34).Nevertheless, White and his coworkers then reported that when platelets in an EDTA-containing buffer were stimulated by thrombin and analyzed by flow cytometry, the binding of MoAbs to Gp Ib decreased by 60-8070 (35) (also see below).…”

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confidence: 86%

Bidirectional Trafficking of Membrane Glycoproteins following Platelet Activation in Suspension

Nurden

1997

Thromb Haemost

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!ntroduction ma membrane permits platelets to interact with leukocytes at sites of vessel injury (10). The fact that many membrane glycoproteins are common to the plasma membrane, SCCS and granule membranes (13), also means that secretion can lead to an increased number of receptors at the platelet surface. Yet, parallel alternative pathways clear surface receptors to the internal membrane systems of platelets and this is how the concept of "bidirectional trafficking" has been brought about. The laffer process has been reported for GP Ib-X-V complexes and ligandbound GP IIb-IIIa complexes. It has been implied to be pafi of a downregulation mechanism for the seven transmembrane domain thrombin receptor (14). Fig. I summarizes some of the events that form the basis of this review as I focalize on the movements of membrane glycoproteins within the membrane systems of platelets activated in suspension. Platelet adhesion and aggregation involve membrane glycoproteins (GPs) that act as specific receptors for ligands mediating platelet-extracellular matrix or platelet-platelet interactions (1). Thg fact that platelets circulate without adhering to each other or to the undamaged vessel wall results from the special properties that govern the interactions between the receptors and their ligands. Adhesion transforms circulating platelets into immobilized cells attached to subendothelial components. GP h-IX complexes promote adhesion by binding to von Willebrand factor (vWF), yet vWF multimers in plasma do not, in a normal situation, express the conformation recognized by the receptor. To acquire this conformation, vWF must be attached to a surface (i.e. the subendothelial matrix) or be submitted to high shear forces (2). Platelet receptors to collagen as well as surface glycoproteins reacting with fibronectin, laminin or thrombospondin also bind immediately when they come into contact with their ligand exposed in subendothelium (data reviewed in 1). In conffast, GP IIb-IIIa complexes, which mediate platelet aggregation, bind soluble fibrinogen, vWF, or other adhesive proteins only after the receptor has undergone a conformational change consequent to platelet activation. This review presents the concept that platelets also modulate their surface properties through the activation-dependent redistribution of receptors between different membrane compartments. As described by White (3), the plasma membrane of the resting platelet invaginates into the cytoplasm so forming an intricate network of channels termed the surface-connected canalicular system or open canalicular system (SCCS or OCS). This is to be distinguished from the membranes of the dense tubular system and those of the secretory granules. During secretion, the general concept is that storage organelles fuse to form secretory vacuoles (4-6) although dense granules have been reported to empty their contents directly at the platelet surface (7). In the former situation, granule contents pass to the external medium by way of the channels of the SCCS. Secretion provide...

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Retention of glycoprotein Ib/IX receptors on external surfaces of thrombin-activated platelets in suspension

Cited by 19 publications

References 14 publications

Glycoprotein Ib Is Homogeneously Distributed on External and Internal Membranes of Resting Platelets

Glycoprotein Ib Is Homogeneously Distributed on External and Internal Membranes of Resting Platelets

Redistribution of glycoprotein Ib within platelets in response to protease-activated receptors 1 and 4: roles of cytoskeleton and calcium

Bidirectional Trafficking of Membrane Glycoproteins following Platelet Activation in Suspension

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