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.

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.

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.

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.

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.

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.

Alterations in lipid fluidity induced by cholesterol and cholesterol hemisuccinate modulate the organization of microtubule skeletons in epithelial cells.

This study was designed to evaluate the role that cholesterol, and the more hydrophilic ester, cholesterol hemisuccinate (CHS) have on the ability of epithelial cells to attach, spread and reorganize microtubule skeletons on a defined substratum. A431 carcinoma cells were grown and incubated with cholesterol, or CHS in polyvinyl pyrrolidone. Subsequently the cells were plated either on plastic petri dishes or plasticware coated with collagen IV or laminin. The alteration in apparent membrane microviscosity was ascertained using fluorescence polarization measurements. Organization of microtubules was determined by immunofluorescence, and by transmission electron microscopy. Cholesterol and CHS inhibited attachment and spreading of epithelial cells. Cells previously attached and spread became spherical after treatment with cholesterol and CHS, but microtubules were unaffected. However, when the cells were pretreated in suspension with cholesterol or CHS the membrane microviscosities markedly increased, and upon subsequent plating those cells adhering neither spread nor organized microtubule skeletons. These results suggest that cholesterol-induced changes in lipid microviscosity modulate the membrane dynamics that control the ability of epithelial cells to attach, spread and organize microtubule skeletons.

Identification of basement membrane components and intermediate filaments in calcifying epithelial odontogenic tumors.

An immunohistochemical technique was used on paraffin-embedded tissues of four cases of calcifying epithelial odontogenic tumors (CEOT). These studies were performed to gain an additional understanding of the nature of amyloid-like deposits in these tumors. For these studies antibodies to Type IV collagen, laminin, and the five classes of intermediate filament proteins were employed. In all of the tumors examined basement membrane components and intermediate filament proteins (cytokeratin) were demonstrated both in the epithelial tumor islands and within the extracellular amyloid-like deposits. Antibodies to vimentin intermediate filaments were localized only in the stromal fibroblasts. Limited proteolysis or the use of a chaotropic agent was required to express the antigenic determinants present. These studies substantiate the presence of basement membrane components in the amyloid-like deposits of CEOT. In addition, these extracellular deposits are shown to be heterogenous in composition by the immunohistochemical demonstration of cytokeratin intermediate filament proteins.

Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly.

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.

Noncollagenous phosphoprotein derived from teleostean fish-scales.

A noncollagenous phosphoprotein derived from teleostean fish-scales is extracted by the use of a dissociative extraction scheme, utilizing 4 M guanidine-HCl, pH 7.4, followed by a 4 M guanidine-HCl, 0.5 M EDTA, pH 7.4. DEAE-cellulose chromatography and repeated Sepharose CL-6B chromatography are used to purify this protein. The isolated protein contains extensive quantities of aspartic and glutamic acids in addition to O-phosphoserine and O-phosphothreonine. The apparent molecular weight of the phosphoprotein as determined by Sepharose CL-6B chromatography is 13 000.

Electron-microscopical studies of conformational changes in dentinal phosphophoryn.

p67tinal phosphophoryn was isolated and purified from unerupted calves molars by the methods of Butler et al. (1981). The resulting proteins were concurrently analyzed by circular dichroism and electron microscopy after low-angle rotary shadowing. Electron microscopy of these proteins in aqueous solutions revealed extended bead-like chains that possessed intramolecular variations in morphology. The addition of calcium ion or methanol to solutions of these proteins produced circular dichroism spectra indicative of more ordered structures. Electron microscopy of these preparations revealed aggregates of 25-30 nm disc-like structures. Although correlations of domain sequences and structure were not possible, the resulting structures did possess molecular morphologies that are compatible with some of the functional roles advocated for these proteins as calcium hydroxyapatite nucleating sites in the mineralization of dentin (Lechner et al., 1981).

The interaction of bovine dentine phosphophoryn and collagen during fibrillogenesis of collagen in vitro.

Bovine dentinal phosphophoryn retards the rate of collagen self-assembly when monomeric collagen is the kinetic unit in fibrillogenesis in vitro. This inhibition is dependent on phosphorylation of the protein and affects the lag period rather than the growth phase for the formation of collagen fibrils. Treatment of the phosphophoryn with calcium markedly increases the inhibitory effect. The use of several fluorescent hydrophobic probes indicates that the calcium-binding to phosphophoryn does not expose any additional interacting hydrophobic domains, thus suggesting that calcium potentiates this interaction, probably by providing a different spatial arrangement of charged groups on this polyelectrolyte, phosphophoryn.