Marlys D. Krumwiede

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

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.

EDTA induced changes in platelet structure and function: influence on particle uptake.

Ethylenediaminetetraacetic acid (EDTA) causes structural, biochemical and functional damage to blood platelets. The alterations induced are considered irreversible. However, the degree of irreversibility, and whether all functions are similarly compromised by EDTA have not been fully evaluated. The present study has examined platelets treated with EDTA to produce the structural changes in channels of the open canalicular system (OCS) associated with irreversible dissociation of the fibrinogen receptor, GPIIb-IIIa (alpha(IIb)beta(3)), for their ability to interact with particulates in suspension. Despite severe narrowing and near occlusion of peripherally oriented OCS channels by EDTA, treated cells were able to bind, translocate and take up fibrinogen-coated gold particles (Fgn/Au), colloidal gold and latex spheres. Thus exposure to EDTA may compromise some aspects of platelet function, but not others which may be important for participation in hemostatic events.

Influence of combined thrombin stimulation, surface activation, and receptor occupancy on organization of GPIb/IX receptors on human platelets.

Summary. Down‐regulation and clearance of as many as 60–80% of GPIb/IX receptors from exposed surfaces on thrombin‐activated platelets to channels of the open canalicular system (OCS) is considered to be a fundamental mechanism regulating platelet adhesivity in vitro and in vivo. The present study has combined thrombin stimulation in suspension, surface activation on formvar grids, receptor occupancy by von Willebrand factor (vWF) and exposure to anti‐vWF antibody in an effort to demonstrate the removal of GPIb/IX receptors from activated cells. Individually the stimuli failed to cause any change in the frequency of GPIb/ IX receptors. Combined, the stimuli were no more effective than when each was used alone. The only way to cause GPIb/IX to move was to add anti‐vWF to thrombin‐activated platelets allowed to spread on formvar grids and covered with multimers of ristocetin‐activated human or bovine vWF. Translocation of GPIb/IX‐vWF‐anti‐vWF complexes from peripheral margins into caps over cell centres, however, did not clear the peripheral zone of vWF binding capacity. Exposure of capped platelets after fixation to a second incubation with vWF demonstrated as many multimers extending from the central cap to the peripheral margins as were seen on platelets exposed a single time to vWF. Antibodies to GPIb, but not to GPIIb/IIIA, prevented the second labelling by vWF. Down‐regulation or clearance of GPIb/IX, in light of this study, does not appear to be a fundamental mechanism modulating platelet adhesivity.

Redistribution of GPIb/IX and GPIIb/IIIa during spreading of discoid platelets

Summary. The purpose of the present study was to prelabel mobile receptors on discoid platelets with specific ligands identifiable in the electron microscope and follow their redistribution during spreading. Platelets were incubated in suspension with cytochalasin E (CE) to preserve discoid form, chilled and mounted on cold formvar grids or glass slide fragments to inhibit receptor movement, covered with cold bovine or ristocetin‐activated human plasmas as sources of vWF to bind GPIb/IX, fibrinogen‐coated gold particles (Fgn/ Au) to couple GPIIb/IIIa, or both probes simultaneously, washed to remove CE and rewarmed to 37°C for intervals up to 30min to stimulate spreading. After brief fixation grids and glass fragments were incubated with anti‐vWF antibody and, subsequently, staphylococcal protein A coupled to 5 nm and 10 nm gold particles to detect vWF multimers. Virtually all of the CE‐treated chilled platelets retained their discoid shape. Half of the discs (53‐3%) bound Fgn/Au, and all bound vWF. Receptors for both ligands were randomly dispersed on discoid cells from edge to edge. During re warming discoid platelets expanded into spread forms. Fgn/Au‐GPIIb/IIIa complexes moved into caps over cell centres and into residual channels of the open canalicular system (OCS). vWF bound to GPIb/IX moved with the cell membrane as the surface expanded during spreading. Discoid platelets prelabelled with both ligands demonstrated similar findings. During rewarming Fgn/Au‐GPIIb/IIIa complexes moved to cell centres and into OCS channels. vWF multimers bound to GPIb/IX moved apart from each other toward peripheral margins of the spread cells. Thus, surface activation resulting in conversion of discoid platelets to spread forms does not cause clearance of GPIb/IX receptors to cell centres and channels of the OCS in the manner that GPIIb/IIIa receptors coupled to Fgn/Au are simultaneously translocated and concentrated in OCS channels.

Normal-sized primary lysosomes are present in Chediak-Higashi syndrome neutrophils.

ABSTRACT. Azurophil granules of normal neutrophils are known to be primary lysosomes and contain myeloperoxidase activity. The present study has used ultrastructural cytochemistry to selectively stain myeloperoxidase containing granules of normal and Chediak-Higashi syndrome neutrophils and serial thin sections to determine if all peroxidase-positive organelles in Chediak-Higashi syndrome cells are protrusions of the huge inclusions characteristic of the disorder. Peroxidase-positive organelles in polymorphonuclear leukocytes from three patients with Chediak-Higashi syndrome varied in size from vesicles and normal-sized lysosomes to the huge bodies filling the cytoplasm. Serial sections demonstrated that the small and normal-sized organelles were commonly present and independent of the giant granules. One type of the normalsized lysosomes contained small vesicles free of peroxidase activity, conveying a honeycomb-like appearance to the matrix. This variety, in particular, was identified in various stages of fusion with giant organelles in circulating Chediak-Higashi syndrome neutrophils.

Induction of GPIb/IX-vWF Receptor-Ligand Translocation on Surface-Activated Platelets

Multimers of von Willebrand factor (vWF) readily bind to glycoprotein (GP) Ib/IX receptors on spread human platelets and cover the cell from edge to edge. Addition of anti-vWF antibody to spread platelets covered with vWF caused the multimers to move from peripheral margins into caps over platelet centers. Despite almost complete centralization of receptor-ligand complexes, a significant number of GPIb/IX receptors capable of binding multimers remained available on the peripheral zone. Fixation followed by a second incubation with vWF, anti-vWF, and staph protein A coupled to 5-nm gold particles (PAG5) revealed multimers extending from the centrally concentrated cap of vWF to cell margins. If spread platelets with central caps of vWF were exposed a second time to multimers and anti-vWF antibody before fixation and stained with PAG5 after, the residual GPIb/IX receptors and second wave of vWF formed a ring around the cap, leaving a clear margin. If after fixation and staining with PAG5 the grids with caps and rings of vWF were washed, exposed a third time to vWF, refixed, and then incubated with anti-vWF and PAG10, the clear margin was covered with multimers of vWF forming a second ring around the first circle of receptor-ligand complexes. Thin sections of spread platelets with central caps of GPIb/IX-vWF complexes revealed only rare examples of uptake by the open canalicular system. The interaction of GPIb/IX with vWF multimers observed in the present study suggests a mechanism by which platelets under high shear forces may adhere and attach firmly to a denuded vascular surface.

Uptake of vWF–Anti-vWF Complexes by Platelets in Suspension

Efforts to identify the translocation of glycoprotein (GP) Ib/IX receptors, either bound to von Willebrand factor (vWF) or not, from exposed surfaces to interior membranes of thrombin-activated platelets in suspension have been unsuccessful. To observe vWF uptake by platelets, we added an anti-vWF antibody and staphylococcal protein A-gold (to act as a marker for the antibody) to an incubation medium containing washed platelets and bovine plasma vWF or ristocetin-activated human vWF. Thin sections of platelets incubated for 10, 20, or 30 minutes with vWF but without antibody revealed no internalization and minimal changes in the original discoid form. Over the same 30-minute period with anti-vWF, however, GPIb/IX-vWF-anti-vWF complexes were cleared from cell exteriors to channels of the open canalicular system. Engorgement of the open canalicular system with vWF multimers resulted in changes in shape, internal transformation, and degranulation. Physical changes associated with anti-vWF-induced uptake of vWF are not seen in platelets that are involved in hemostatic plug formation or clot retraction.

Localization of GPIb/IX and GPIIb/IIIa on Discoid Platelets.

The organization and reorganization of mobile receptors, GPIIb/IIIa and GPIb/IX, on surface- and suspension-activated platelets have been studied in detail, but their distribution on resting, discoid platelets is uncertain. The present study has treated platelets in suspension with cytochalasin E before mounting on formvar grids or glass slide fragments in order to preserve their discoid appearance, then probed the organization of GPIIb/IIIa with fibrinogen coupled to gold particles (Fgn/Au) and GPIb/IX with bovine or ristocetin-activated human plasma detected by combined anti-vWF antibody and protein A coupled to gold particles. Multimers of vWF had the same tortuous, linear distribution from edge to edge observed previously on surface-activated platelets. However, the gold particles marking the complex of vWF-anti-vWF bound to GPIb/M were closer together on the discoid cells. Fgn/Au particles bound to GPIIb/IIIa receptors were uniformly distributed from edge to edge on many discoid platelets. On others they tended to clump or cluster in strips or patches. The latter organization of Fgn/Au-GPIIb/IIIa receptors may be due to the rugose nature of the discoid platelet surface or an influence of cytochalasin E. Definition of mobile receptor organization on discoid cells provides a useful baseline for determining their fate following surface or suspension activation.

Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets

Recent reports have suggested that spike-like pseudopods develop on platelets loaded with intracellular chelating agents during glass activation, and proposed that the extensions are caused by an unusual organization of newly assembled actin filaments. The present study has reexamined this hypothesis. Platelets loaded with one of the chelating agents, Quin II or BAPTA, seldom formed spike-like pseudopods when exposed to glass at 37 C for 30-60 min. However, when chilled and rewarmed the Quin II-or BAPTA-loaded platelets readily developed one or several spike-like extensions after a 30-60-min exposure to glass or on shaking in suspension. Thin section and negative-stain electron microscopy demonstrated that the major constituents of spike-like pseudopods were microtubules. Unusual coils of actin filaments were not observed. The observation was confirmed by immunofluorescence microscopy employing an anti-tubulin antibody and fluorescein-conjugated antimouse IgG. Cytochalasin B, an agent that inhibits new actin filament formation had virtually no effect on spike formation by chilled-rewarmed, Quin II- or BAPTA-loaded cells, whereas prior exposure to vincristine, an agent that dis-assembles microtubules and prevents their reformation, blocked spike development. Taxol, a drug that stabilizes microtubules and prevents their disassembly by cold or vincristine, prevented spike formation. Results indicate that microtubule assembly is the major cause of spike-like pseudo-pod formation, and the increased assembly may be due to binding of free cytoplasmic calcium by intracellular chelating agents.

Platelet particle processing: an example of surface sorting on single cells.

The ability of glycoprotein IIb-IIIa (GPIIb-IIIa) receptors on fully spread, surface-activated platelets to bind specific antibodies or ligands, singly or sequentially, and clear them toward cell centres has been established in several investigations. However, the basic mechanism involved in receptor-ligand translocation remains unclear. The present study has attempted to provide additional information by adding two different electron-dense probes simultaneously to surface-activated cells. Over a 5 min period of incubation small latex particles were cleared more rapidly from platelet margins to cell centres than simultaneously added particles of colloidal gold coupled to fibrinogen (Fgn/Au). Large and small latex spherules mixed together and added to spread platelets under the same conditions were moved at the same rate and concentrated together in the cell centres. Results of this investigation indicate that simple diffusion is unlikely to be the generating force for movement of receptor complexes on platelet plasma membranes.