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We studied megakaryocyte processes formed in rat bone marrow and spleen, using both the transmission and scanning electron microscopes. Some processes were bulky, others slender and beaded. The bulky megakaryocyte processes developed a specialized arrangement of organelles at the site at which they entered the lumen: filaments present around the outside of the process seemed to support a central cylinder in which organelles flowed along microtubules. Megakaryocyte processes were present in platelet-rich plasma from both human and rat blood. When followed in living preparations, bulky processes developed pointed tips, elongated, and became slender and beaded. Fusiform proplatelets also were present in the platelet rich plasma, with pointed tips at both ends of what appeared to be single "beads"; we assume that the long, beaded megakaryocyte processes would have fragmented were we to have had proper culture conditions. The straight, shorter fusiform proplatelets in living preparations underwent characteristic curving and bending motions, eventually forming disk-shaped cells which sometimes had appendages. This behaviour suggests that the entire process of platelet morphogenesis takes place in plasma: megakaryocyte processes first elongate, then bead and fragment, and then curve and fuse to form disk-shaped platelets. This interpretation is strengthened by finding in freshly isolated plasma many of the shapes seen in the transformations studied in living cell preparations. The megakaryocyte processes and the proplatelets seemed to appear in plasma with a periodicity related to light and dark cycles--that is, with a circadian rhythm. In particular, megakaryocyte processes appear in human blood within a few hours after sunrise; we argue that this might be related to similar peak periods for heart attacks.

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Certain peptides with sequences related to part of the major histocompatibility complex class I antigen enhance the action of insulin. These peptides also aggregate into fibrous structures that seem to be related to their biological activity. In the current study, the 17-residue peptide with amino acid sequence Gly-Asn-Glu-Gln-Ser-Phe-Arg-Val-Asp-Leu-Arg-Thr-Leu-Leu-Arg-Tyr-Ala is used as a representative example of these bioactive molecules. As seen by electron microscopy, the peptide associates into gently twisted ribbons, 50 A thick, in which the amount of twist decreases as the ribbons become wider. X-ray diffraction analysis suggests that the peptides are arranged as in an antiparallel beta-sheet extending essentially endlessly along the fiber axis. The amino acid sequence of the peptide is such that one side of the beta-sheet is predominantly polar while the opposite side is nonpolar. This allows the beta-sheets to form multilayers with alternating hydrophobic and hydrophilic interfaces. The length of the extended peptide (approximately 54 A) determines the thickness of the ribbon and the tendency of individual beta-sheets to twist accounts for the twisting of the ribbons. An alternative model is also discussed, again based on antiparallel beta-sheets, but with adjacent sheets interdigitated in a "side-by-side" fashion rather than forming stacked layers. Comparable inactive peptides such as Gly-Asn-Glu-Gln-Ser-Ala-Arg-Val-Asp-Leu-Arg-Thr-Leu-Leu-Arg-Tyr-Tyr (changed amino acids underlined) do not form ordered filamentous structures.

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The platelet population in man and rat can be divided into two classes of about equal size based on the presence/absence of a p-nitrophenylphosphatase, which probably is a phosphotyrosine phosphatase (PTPase). Phosphorylation of tyrosines on several platelet proteins is implicated in platelet activation, and I carried out in vitro and in vivo experiments on rats to determine whether PTPase positive and negative platelets differed in their reaction time. I used adhesion to collagen in vitro and in vivo (longitudinal slits in aorta and vena portae) and platelet aggregates in clots formed in vivo. I present evidence that PTPase negative platelets react the fastest, most conspicuously seen in the arterial bleeding under high flow conditions, where the first platelets to respond and adhere are predominantly PTPase negative.

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The platelet population of man and rat can be divided into two classes of about equal size on the basis of presence/absence of an acid phosphatase which acts on para-nitrophenylphosphate (a PNPase), at pH 5. The cytochemical reaction product is in the platelet cytoplasmic matrix, without apparent association with organelles or membrane systems. We could not relate differences in staining to differences in function: all cells responded the same to activation by thrombin, ADP, or collagen, in fibrinogen binding to activated platelets, by endocytosis of fluid-phase tracers, and in internalization of latex particles. With respect to possible physiological substrates for the PNP-ase, there was no reaction product from beta-glycerophosphate, AMP, ADP, ATP, GTP, CMP, IMP, cAMP, creatine phosphate, and inositol phosphates, and the enzyme was not inhibited by 40 mM lithium. There was reaction product from tyrosine phosphate suggesting that the physiological substrate for PNP-ase is tyrosine phosphate. In rat bone marrow, megakaryocytes also were of two classes, PNPase positive and PNPase negative, suggesting that different classes of platelets arise from different classes of megakaryocytes.

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Blood platelets are produced in the circulation by fragmentation of long, slender processes of cytoplasm formed from the megakaryocyte, the parent cell of platelets. Fragmentation occurs at local constrictions, forming 6 to 15 µm long, fusiform fragments (elongated proplatelets). The fusiforms transform into the circular, disc-shaped mature platelet by curving into a ring, which closes by fusion at the tips. The hole in the ring is finally filled in by a centripetal flow of membrane from the periphery. It is presumed that the curving of the fusiform is mediated by curving of its contained bundle of microtubules, which becomes the marginal bundle of the disc-shaped platelet. When curving begins in the fusiform, microtubules are closely associated with a membraneous tubule that becomes the submarginal tubule of the dense tubular system.

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A morphological study of the endocytic pathways in non-activated human blood platelets has been made using fibrinogen-stabilized colloidal gold as a fluid-phase marker, and acid phosphatase as a marker for lysosomes. Two pathways of constitutive endocytosis were demonstrated: one pathway was mediated by clathrin coated vesicles which formed at the platelet surface and the surface-connected, canalicular membrane system and which fuse with and deliver their contents to the platelets' secretory alpha-granules. The other pathway follows a degrading, endosomal-lysosomal route and appears to be clathrin independent. Endosomes containing marker particles originate from the surface-connected canalicular system, through which ambient fluid percolates. An account is given of the pleomorphism of platelet lysosomes, and evidence is presented for a lysosomal, focal autophagia in platelets which may be linked to platelet senescence. Lysosomes in non-activated platelets are thus involved in the same processes as lysosomes in other cells.

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Platelet function and morphology were studied in eight healthy male volunteers before, immediately after, and 1 d after the infusion of 250 mL of 10% Intralipid. The plasma concentrations of the platelet-release products beta-thromboglobulin (beta-TG), serotonin (5-HT), and platelet factor 4 (PF4) and the threshold to ADP- and adrenaline-induced aggregation were determined ex vivo. In addition the platelets were examined by electron microscopy. Although platelets released beta-TG, PF4, and 5-HT after the infusion, there was no significant change in ex vivo aggregability. About 3% of the platelets had internalized small-sized lipid particles (0.1-0.2 micron) whereas no other morphological changes were detected. The release products may negatively affect the hemorheological properties of the microcirculation in critically ill patients. The release of PF4 may explain the antiheparin effect of parenteral lipid therapy.

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Electron microscopy of mammalian blood platelets after contrast enhancement with tannic acid after an initial fixation in glutaraldehyde and osmium reveals numerous coated pits (c.p.) and vesicles (c.v.), indicating a process of receptor-mediated endocytosis. The c.p. may be located at any site of the plasma membrane or the canalicular, surface connected membrane system. C.v. fuse with platelet granules without losing their coat. Evidence for a continuous transfer of ambient fluid to granules via c.p. and c.v. was obtained by the use of fluid-phase markers. It is proposed that the endocytic process may play a role in blood platelet activation.

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When human blood platelets spread on a substratum they increase their surface area as much as 4-fold. We investigated the mechanism of spreading by light microscopy and by scanning and transmission electron microscopy. Contact of a platelet with a glass surface induces formation of thin extensions which spread out over the substratum. These extensions resemble the actin-containing microspikes and lammelipodia of tissue cells in culture and appear to be drawn from the peripheral cortical layer associated with the plasma membrane. If platelets are initially labeled on their external surface with cationic ferritin or lentil-conjugated gold particles and then allowed to spread, the labels are retained in the central region, or granulomere. Proteins released by the spreading platelet--fibronectin and fibrinogen--also remain in this central unspread region. Peripheral regions of spread platelet surface (hyalomere) were unlabeled following the above procedures but could be labeled with cationic ferritin or lentil-conjugated gold provided these were applied after spreading was completed. These markers are cleared with time from the periphery, moving centripetally to accumulate at the granulomere. We suggest, on the basis of these observations, that platelets spread onto a substratum by a closely similar mechanism to that used by cells such as fibroblasts. In both cases the spreading involves the peripheral actin cortex and is accompanied by a continual centripetal movement of surface components--a "membrane flow"--which continues even after spreading is completed.

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Adhesion of rat blood platelets to native rat tail collagen fibrils was studied in the electron microscope under conditions that preserved collagen-associated proteoglycans (CAPG). The CAPG molecules were aligned in chain-like configurations that encircled the fibrils with a 65 nm period; they appeared to coat the fibrils completely and extended 60-100 nm away from the fibril. The initial platelet-fibril contact occurred between the platelet glycocalyx and the CAPG of the fibrils i.e. between two surfaces with net-negative charges. When close contact was established between the fibril surface proper and the platelet membrane, CAPG were not identified in the area of contact, and the collagen-platelet distance was reduced to a approximately 10-12 nm wide gap traversed by delicate links in register with fibril periodicities.

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Human blood platelets were challenged sequentially in vitro with polyclonal anti-platelet antibodies and cationized ferritin. Both ligands bound to the surface membrane and were sequestered by internalization into a surface-connected membrane system (SCS) with a cleared surface membrane as the eventual result. Patching and capping of bound antibody preceded internalization, and platelets cooperated in the clearing process by adhering to each other at capped areas and by mutual covering up, a process dubbed platelet hugging. The internalization process was repeated upon challenge with the second ligand, the two ligands being sequestered as separate deposits in the SCS, mirroring the two cycles of internalization. Platelets were activated during internalization process and tended to aggregate. In aggregates, surfaces exposed to the medium were cleared of ligand, which accumulated in the intercellular spaces and within the SCS of the aggregated platelets. Aggregation but not internalization and hugging was prevented by adenosine and adenosine monophosphate.

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Colloidal gold solutions conjugated with staphylococcal protein A (SpA) are widely used in high-resolution immunocytochemical studies to visualize antibodies bound at antigenic sites. Here we report that colloidal gold solutions conjugated with SpA, bovine serum albumin (BSA), or gelatin bind selectively to structures in glutaraldehyde-fixed, plastic-embedded epidermis of rabbit, mouse, and human. Two types of keratohyaline granules are present in epidermis, a phosphorus-rich (PR) and a sulphur-rich (SR) type. The PR keratohyaline granules were strongly labeled with gold particles, whereas SR keratohyaline granules or other structures in the living cells of epidermis were unlabeled. The PR keratohyaline granules are assumed to be precursors of the matrix protein of cornified cells, and intense gold labeling occurred over the lower layer of cornified cells (i.e., stratum lucidum). More superficial cornified cells were weakly labeled or unlabeled. The gold labeling pattern was identical whether SpA, BSA, or gelatin was used to stabilize the colloidal gold solution. The mechanism of binding of protein-conjugated gold to PR keratohyaline granules and matrix protein of cornified cells is not clear. It is speculated that the charged gold particles are not completely coated by the stabilizing protein, allowing for an electrostatic interaction with charged proteins in sections of cells.

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Phenotypic characteristics of a cloned cell line, RH-SLC-L11, established from a human squamous lung carcinoma, were studied. The line has maintained its morphologically characteristic growth pattern for over 3 years. Settling cells exhibited extensive surface blebbing during spreading and established small cell islands that eventually expanded by mitosis to confluent cultures. Cell islands and confluent cultures presented three cell types: (i) small, polygonal cells, (ii) polygonal cells of intermediary size and (iii) very large, extremely flattened, degenerating cells. Mitotic activity was present predominantly in type (i) and the sequence (i)--(iii) is presumed to represent the lines' cycle. Previous work has demonstrated that the SLC-L11 line releases tumor-associated glycoproteins and glycolipids. These could be identified with a murine Mab (43-9F). The specific epitope was determined by carbohydrate residues and was shown to have growth factor-like properties. Mab 43-9F bound heterogeneously to the surface of SLC-L11 cells: Most large cells were unreactive while both type (i) and (ii) cells showed conspicuous differences in immunostaining intensity. Immunocytochemical analysis also indicated redistribution, shedding and internalization of antigen-Mab complexes, which may have significant impact on the use of the epitope as tumor marker in diagnosis and therapy. No definite clue was obtained as to the release of the antigenic carbohydrate epitope itself.

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Filipin, a complex of polyene antibiotics, forms morphologically distinctive complexes with cholesterol in cell membranes under proper experimental conditions. When applied to non-activated, discoid platelets, filipin-induced lesions (FIL) occurred in rows at the platelet equator, suggesting a specialized membrane organization at the platelets' largest circumference. In some thrombin-activated platelets we observed surface membrane blebbing and release of lipid vesicles that predominantly originated from the plasma membrane proper, but some originated from (unidentified) platelet granules. FIL were initially present in high numbers over the entire bleb, they accumulated later at the neck of blebs, while the released vesicle was free of FIL. Absence of intramembrane protein particles (IMP) from the membranes of blebs and vesicles suggests that released vesicles are essentially without cholesterol and intrinsic membrane proteins and may consist predominantly of phospholipids. Membrane blebbing and vesicle release may represent unmasking and release of procoagulant platelet factor 3 activity.

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The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.

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Cloned human cell lines of squamous-cell lung carcinoma and small-cell lung carcinoma were treated with 5-azacytidine (5-azaC), 12-0-tetradecanoyl-phorbol-13 acetate (TPA), retinoic acid (RA), or a combination of these drugs, and the effects on cellular morphology, in vitro growth properties, antigenicity, tumorigenicity and metastatic activity in nude mice were studied. Antigenicity was measured by the expression of major histocompatibility antigens (MHC) and of lung-tumor-associated antigens. 5-AzaC treatment resulted in subclones with shorter population doubling times (from 40-50 hr down to 14-20 hr) and increased cloning efficiencies (from less than 1% to 5-50%). TPA and RA induced loss of proliferative activity in vitro and tumorigenicity in vivo of both lines. This was morphologically associated with the appearance of an abundance of large vaculated cells which by DNA-analysis were all found to be in G0-G1 phase. However, 2 of the 5-azaC-treated subclones were insensitive to both TPA and RA, whereas the remaining subclones (20) all responded like untreated lines to TPA and RA. One of the sublines insensitive to TPA and RA formed metastases in nude mice in contrast to all the other lines used. The density of lung-tumor-associated antigens was significantly reduced by TPA-RA treatment, whereas the expression of MHC antigens was unaffected. In contrast, 5-azaC resulted in some cases in increased density of MHC antigens. The effects of 5-azaC on cellular phenotypes were not directly correlated to the total genomic content of 5-methylcytosine. The data suggest that this experimental system is suitable for studies of phenotypic features of malignant cells as related to cellular differentiation.

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The cellular origin of the malignant cells in Hodgkin's disease (HD) has been discussed for several decades. Previous investigations on fresh biopsy material and cultured cells from lesions of Hodgkin's disease have led to various suggestions such as a T lymphocyte, B lymphocyte or monocyte/macrophage origin of HD cells. However, all these studies have been hampered by uncertainty in the identification of the truly malignant cells. It has therefore been desirable to establish in vitro lines of the malignant cell population in HD. A number of cell lines from HD lesions has been described, but most of these studies can, upon a critical appraisal, be dismissed as not being representative for Hodgkin's and Reed-Sternberg cells. We report on the phenotypes of established cell lines of Hodgkin's cells and conclude that it is most likely that the malignant Hodgkin's cells are derived from cells of the monocyte/macrophage lineage.

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Filipin, a mixture of polyene antibiotics which form complexes with cholesterol, perturbs membrane lipid organization, and causes hemolysis of erythrocytes, is increasingly used as a cytochemical probe for the distribution of cholesterol in cell membranes. We used light (phase-contrast, dark-field and fluorescence) and electron microscopical techniques (whole-mount shadowing, negative staining, and freeze-fracture) to study the interaction of filipin with unfixed and glutaraldehyde-fixed human red blood cell (RBC) membranes. Lysis time and extent depended upon the cholesterol:filipin (C:F) ratio. Lysis was prevented by osmotic protection with high MW dextran. Filipin treated cells fluoresced, but variation in fluorescence intensity among unfixed as well as among fixed cells was evident both at low and high C:F ratios. Negatively stained preparations of unfixed cells lysed on grids or in suspension revealed ring- or C-shaped filipin-induced lesions (FIL) equipped with a veil-like appendage; single FIL, and FIL fused by their veils into aggregates, were shed from membranes. FIL at the surface proper of shadowed whole-mounts and of freeze-etched preparations of prefixed cells appeared as single, dispersed or aggregated cylinders protruding to variable heights above the membrane's plane; aggregated FIL were shed from cells. The freeze-fracture appearance of FIL differed in membranes fixed before or after filipin treatment. E- and P-faces of post-fixed membranes exhibited cylindrical protrusions and depressions, respectively; in essence, the reverse was found in pre-fixed RBC. Both pre- and post-fixed membranes showed considerable variation in the number of FIL on individual cells whether incubated at high (1:1) or low (1:5) C:F ratios, or for a short (10 min) or a long (80-180 min) time. Aggregation and shedding of FIL was evident in all preparations. Thin layer chromatography of the incubation fluid after sedimentation of cells showed that membrane cholesterol was shed from incubated cells. The presented data question the feasibility of filipin as a probe for the topographical distribution of cholesterol in cell membranes.

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Cloned cell lines and a number of subclones from these lines were established in vitro from biopsies of small cell lung carcinomas and squamous cell lung carcinomas. The cloned cultures, including the cloned subclones, were analyzed in respect to morphology, karyotype, growth rates, clonogenicity in semisolid agar medium, and tumorigenicity in nude mice. A remarkable biologic diversity was found in respect to most of these biologic features. In addition, four murine monoclonal antibodies with high specificity for lung tumor cells were generated. Their reactivity pattern to clonogenic cells was for some clones different as compared to the nonclonogenic cells. Subclones of tumor cells not binding the antibody were identified for each monoclonal antibody. It is concluded that intratumoral phenotypic diversity may have a severe negative impact on the use of monoclonal antibodies in cancer diagnosis/therapy. The work also indicates that a mixture of antibodies may be more useful in tumor diagnosis than individual antibodies and perhaps even therapy, particularly if they bind to the clonogenic part of a cell population.

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