The critical role of myosin IIA in platelet internal contraction.

BACKGROUND: Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined. OBJECTIVE: To define the contribution of myosin IIA to platelet internal contraction. METHODS: Aspirin-treated platelets suspended in calcium-free buffer were activated with a low concentration (25 nm) of the thromboxane A(2) analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently. RESULTS AND CONCLUSIONS: Low-concentration blebbistatin (10 microm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25-100 microm) produced concentration-dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 microm blebbistatin.

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.

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.

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 cytochalasin B (CB) on GP Ib distribution after thrombin or TRAP and before surface activation.

The receptor for von Willebrand factor (vWF) on human platelets, glycoprotein (GP) Ib/IX, has been shown in our studies to be an immobile complex when stimulated in suspension or on surfaces. Recent investigations have revealed that GP Ib/IX remains immobile on platelets activated in suspension followed by exposure to formvar surfaces that cause the cells to spread. However, since channels of the open canalicular system (OCS) are evaginated back on to the exposed surface during spreading, it was suggested that our study missed the clearance of GP Ib/IX from the exposed surface to internal membranes. The present study has added cytochalasin B after exposure of platelets to thrombin or TRAP in suspension in order to prevent spreading and movement of GP Ib/IX during subsequent exposure to surface activation on formvar grids. Results indicate that GP Ib/IX receptors remain randomly dispersed from edge to edge on platelets activated by thrombin or TRAP in suspension 10 minutes before treatment with CB followed by surface activation. Statistical analysis of the frequency of immunogold particles binding to monoclonal antibodies attached to GP Ib/IX revealed no significant reduction in frequency, translocation from cell edges or concentration of GP Ib/IX receptors in or around channels of the OCS. Results support the concept that GP Ib/IX is not cleared from exposed surfaces to the OCS of platelets activated by thrombin or TRAP and surface activation.

Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44.

Fibrosis results when myofibroblasts invade the wound fibrin provisional matrix. Extracellular matrix receptors on the cell surface mediate cell adhesion, migration, and invasion. Recent work with transformed cells indicates that these cells use the cell surface matrix receptor CD44 for migration and invasion. In this study, we examine whether lung fibroblasts, isolated from patients dying with acute alveolar fibrosis, use CD44 to invade a fibrin matrix. Consistent with a role for CD44 in mediating fibroblast invasion and subsequent tissue fibrosis, immunohistochemical analysis of lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue. PCR, Western, and immunoprecipitation analysis demonstrate that the 85-kD CD44 isoform is expressed by acute lung injury fibroblasts. Consistent with a role in mediating matrix adhesion and migration ultrastructurally, CD44 was found uniformly over the cell surface and was found densely labeling filopodia and lamellipodia, highly motile structures involved in cell migration. To determine if lung injury fibroblasts use CD44 to invade fibrin, a fibrin gel model of fibrosis was used. By blocking the function of CD44 with monoclonal antibodies, fibroblast invasion into a fibrin matrix was inhibited. To examine the mechanism by which CD44 mediates fibroblast invasion, the role of CD44 in fibroblast migration and adhesion was evaluated. Anti-CD44 antibody blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and hyaluronic acid. Additionally, fibroblast CD44 mediated adhesion to the provisional matrix proteins fibronectin, fibrin, and hyaluronic acid, but not to laminin, a component of the basement membrane. These findings support the hypothesis that fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue.

Prelabeled glycoprotein Ib/IX receptors are not cleared from exposed surfaces of thrombin-activated platelets.

The present investigation has re-examined the hypothesis proposing that glycoprotein (GP)Ib/IX receptors for von Willebrand factor are rapidly cleared from exposed surfaces to internal membrane systems after activation of platelets by thrombin in suspension. Platelets were prelabeled with either a polyclonal antibody to GPIb alpha, antiglycocalicin (A-Gl), or a cocktail of two monoclonal antibodies, AP1 and 6D1, exposed to 0.1 or 0.2 U/ml thrombin for 5 or 10 minutes, fixed and stained with Staphylococcus protein A coupled to gold to detect A-Gl or goat anti-mouse IgG bound to gold particles to locate AP1 and 6D1 before or after preparation of frozen thin sections or embedding for plastic thin sections. The frequency and distribution of protein-A-gold markers for GPIb/IX on thrombin-activated platelets viewed in thin plastic sections did not differ from the density on resting platelets stained with A-Gl. Cryosections of A-Gl-prelabeled platelets labeled again on cryosections revealed GPIb present on linings of the open canalicular system of resting and activated platelets, but the density of gold in interior channels and frequency of gold particles on exterior surfaces were not altered by thrombin stimulation. Platelets prelabeled with the cocktail of 6D1 and AP1 and studied in cryosections also failed to reveal uptake of GPIb/IX receptors into the open canalicular system after activation by thrombin. The findings do not support the concept that thrombin causes clearance of GPIb/IX receptors from exterior surfaces to interior membranes of activated platelets.

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.

HOCl effects on the tight junctions of rabbit tracheal epithelium.

We previously found that HOCl, produced from neutrophil products in infected airways, decreases electrical resistance (R) of rabbit tracheal epithelium. Interestingly, HOCl at 6 mM, a reasonable concentration in diseased airways, decreased R without apparent cell damage. This study sought to determine whether this noncytotoxic dose of HOCl causes morphological changes that correlate with the decrease in R. Excised rabbit tracheas were treated with 6 mM HOCl for 15 min, before and after which epithelial R was determined. Epithelial tissue was then fixed and prepared for transmission electron microscopy, immunofluorescent labeling of F-actin or the tight junction protein ZO-1, or freeze fracture to examine tight junction strands. HOCl treatment caused a 50% decrease in R. Electron micrographs showed no cell, cell membrane, or tight junction changes. By laser confocal microscopy, 6 mM HOCl did not affect the distribution of F-actin or ZO-1. However, morphometric analysis of freeze-fracture replicas showed that tight junction strand number was significantly decreased from 7.06 +/- 0.09 to 4.79 +/- 0.11 and junctional width was significantly decreased from 0.306 +/- 0.007 to 0.214 +/- 0.006 microns. These latter changes may have contributed to the observed decrease in epithelial R.

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

The present study has evaluated the hypothesis stating that glycoprotein (GP) Ib/IX, the receptor for von Willebrand factor (vWF), is downregulated and cleared from exposed surfaces to channels of the open canalicular system (OCS) on platelets activated by thrombin in suspension. Cryosections of resting and thrombin-activated platelets fixed at intervals of 1 to 30 minutes after stimulation by thrombin and stained with antiglycocalicin antibody and protein A gold showed no decrease in the density of GPIb/IX receptors on the platelet surface or increase on linings of the OCS at any interval after stimulation by thrombin. Thin sections of platelets exposed to thrombin in suspension followed by settling onto a plastic chamber for intervals of 1 to 30 minutes revealed retention of GPIb/IX receptors on exposed surfaces detected by vWF, anti-vWF, and protein A gold throughout the 30-minute period of study. Results of this investigation indicate that GPIb/IX receptors remain on the surface of platelets activated by thrombin in suspension, are not cleared to the OCS, and retain the ability to bind vWF for at least 30 minutes.

Dynamic redistribution of glycoprotein Ib/IX on surface-activated platelets. A second look.

The present study has re-evaluated the mobility of glycoprotein Ib/IX (GPIb/IX), the von Willebrand factor receptor, on surface-activated platelets. A previous report employing immunogold cytochemistry with monoclonal and polyclonal antibodies specific for GPIb/IX concluded that the receptor remained stabilized in plasma membranes and did not move during platelet attachment and spreading on formvar grids, despite the observation that immunogold particles marking GPIb/IX were missing from peripheral margins and pseudopods of the surface-activated platelets. Addition of thrombin to surface-activated, spread platelets freed GPIb/IX from its anchor to the membrane and stimulated movement of receptor-ligand complexes into caps over centers of spread platelets. In our investigation, surface-activated platelets, stimulated or not by thrombin, were fixed in a higher concentration of glutaraldehyde than used by the earlier workers before exposure to monoclonal or polyclonal antibody to GPIb/IX, after incubation with the antibody, but before treatment with the immunogold marker, protein A gold (PAG), or after both antibody and PAG. When fixed before exposure to antibody and PAG, GPIb/IX receptors were dispersed evenly over dendritic and spread platelets from edge to edge, including peripheral margins and pseudopods. Thrombin had no influence on distribution of the receptors. Exposure to antiglycocalicin antibody before fixation caused movement of GPIb/IX receptors from peripheral margins of spread cells and pseudopods of dendritic forms. Thrombin treatment did not enhance the movement. Fixation after exposure of surface-activated platelets, treated or not with thrombin, to antibody and PAG caused movement of GPIb/IX receptors into caps over cell centers. Results indicate that central movement of GPIb/IX receptors is unrelated to surface activation, spreading, or thrombin stimulation. Rather, the translocation is caused by the antiglycocalicin antibody and accentuated by PAG.

A gene causing Hermansky-Pudlak syndrome in a Puerto Rican population maps to chromosome 10q2.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder that affects pigment production and platelet function and causes the deposition of a ceroid-like material in various tissues. Variability in the phenotype and the presence of several potential mouse models suggest that HPS may be a heterogeneous disorder. In order to identify a gene responsible for HPS, we collected blood samples from a relatively homogeneous population in Puerto Rico where the HPS carrier frequency is estimated to be 1 in 21. Analysis of pooled DNA samples allowed us to rapidly screen the genome for candidate loci, and significant evidence for linkage was detected for a marker on chromosome 10q. This region of the human genome is conserved syntenically with the region on mouse chromosome 19 where two possible mouse models for HPS, pale ear and ruby eye, are located. This linkage result was verified with additional markers, and a maximum LOD score of 5.07 at theta = .001 was calculated for marker D10S198. Haplotype analysis places the HPS gene in a region of approximately 14 cM that contains the markers D10S198 and D10S1239.

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

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 degrees C for intervals up to 30 min 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 rewarming 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.

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.

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.

The down-side of platelet interaction with surfaces.

Events occurring on the exposed luminal surface of blood platelets during interaction with foreign surfaces have been evaluated in detail, but the down-side has received less attention. The present study has examined the interaction of platelets with glass precoated with fibrinogen-gold (Fgn/Au), latex spherules, or cationized ferritin (CF) and compared their organization on the ventral surface with the fate of the same particulates when added to platelets already spread on glass or formvar grids. Platelets spread readily on the particle-coated glass surface. During early stages of interaction, particles of Fgn/Au, CF and latex were taken up by channels of the open canalicular system (OCS). Fgn/Au and CF often reached the dorsal surface through OCS channels where they were moved to platelet centers. In contrast, layers of Fgn/Au, CF and latex remained evenly dispersed from edge to edge on the down-side of spread platelets. Clearance of particulates from peripheral and intermediate to central zones under spread platelets was not observed. Particulates did not move from the up-side around platelet margins to the down-side, or from the ventral surface around edges to the up-side, unless spread cells were in contact. Thus, particulates use the OCS as a two-way street. The persistence of evenly dispersed layers of Fgn/Au, CF or latex on precoated glass under spread or spreading platelets indicates a significant difference in the way particulates are handled on the free upside and bound down-side. The results support the concept that the main purpose of mobile receptors is to facilitate spreading, and that the down-side is a protected zone of adhesion.

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

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.

Synergistic effect of storage pool deficient platelets and low plasma von Willebrand factor on the severity of the hemorrhagic diathesis in Hermansky-Pudlak syndrome.

A study of 565 Puerto Rican patients with storage pool deficient (SPD) Hermansky-Pudlak syndrome (HPS) demonstrated that most HPS patients had minor bleeding episodes while others had repeated, severe hemorrhagic episodes requiring transfusion. The severity of bleeding in these latter patients could not be attributed to their SPD alone. As swine with SPD platelets and low von Willebrand factor antigen (vWF:Ag) have more severe hemorrhages than pigs with either defect alone, 146 albino patients and 46 normally pigmented patients were examined for their level of vWF:Ag. The risk of SPD HPS patients having severe, repeated bleeding episodes increased when vWF:Ag fell below 70 U/dL. Family studies indicated that low vWF:Ag levels were more frequently associated with O blood group than from a gene suppressing production or release of vWF1. HPS patients should be tested for vWF:Ag levels.

Role of actin in platelet function.

Cytochalasin E (CE) was used in this study to evaluate relationships between platelet shape change, pseudopod extension, internal transformation, actin filament assembly, fibrinogen receptor expression, aggregation, clot tension and secretion. Various ratios of G- to F-actin, measured by the DNase inhibition assay, were achieved by using several concentrations of CE in stirred and unstirred thrombin-stimulated platelets. An increased rate of actin polymerization was found in "aggregating" conditions as compared to "activating" conditions, the latter achieved by either absence of stirring or stirring in the presence of the tetrapeptide Arg-Gly-Asp-Ser (RGDS). Larger concentrations of CE were required in aggregated than in activated platelets to obtain G-actin levels analogous to those in resting platelets. Concentrations of polymerizing actin above 20% and 55% for activated and aggregated platelets, respectively, trigger platelet shape change and the extension of pseudopods. Prevention of de novo actin polymerization by CE, added either before or after thrombin stimulation, inhibits fibrinogen binding to platelets. This suggests an involvement of the new actin polymers in fibrinogen receptor exposure. As expected, platelet aggregation and clot tension, which are dependent on fibrinogen receptor exposure, were inhibited by CE. Granular secretion was not dependent on polymerizing actin.

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.