Cytoplasmic microtubules in rabbit platelets

Summary A system of microtubules 200–250 Å in diameter is described in the cytoplasm of rabbit platelets. In thin sections they are seen most frequently cut in cross section and at opposite ends of the elliptical platelet where they form a compact bundle containing 10 to 30 microtubules. In other platelet sections they describe a circumferential course 500 to 600 Å deep to the plasma membrane. Occasional microtubules pass between the marginal band and the centre of the platelet.The relationship of the microtubule system to fibres previously observed in the hyaloplasm of platelets spread on films is discussed, and the marginal band of microtubules in platelets equated to that present in nucleated erythrocytes.I am indebted to Dr. E. H. Mercer for his helpful criticisms and to Miss N. Carroll and Mr. R. G. Hill for technical assistance.     

Morphology of rhinoceros platelets

Basic hematological information about rhinoceros species is limited. In this study, we have determined the platelet counts and described the platelet morphology of free-living white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros using light and electron microscopy. The platelet counts of the two species were similar, but there were significant differences in platelet size as well as morphology between the species. The presence of large, nondiscoid platelet forms, resembling proplatelet or stress platelet forms, were observed in both, although not as often in the white rhinoceros as in the black rhinoceros.     

Melatonin synthesis by rabbit platelets

Platelets of reserpinized rabbits, incubated in buffer containing tritiated 5-hydroxytryptamine (³H-5HT), have the ability to convert ³H-5HT into labelled compound (s) extractable with alkaline-chloroform. The bulk of the chloroform-extractable radioactivity showed a Rf value similar to authentic melatonin on silicagel thin-layer chromatography.The labelled product eluted with ethanol from the silicagel area where melatonin was suspected to reside was indentified as melatonin by mass spectrometry and radioimmunoassay.Our in vitro experiments demonstrate that rabbit platelets are capable of converting 5-HT into melatonin apparently because they possess the enzymatic equipment necessary for this biosynthesis i.e. serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase.     

Phospholipid-deacylating enzymes of rabbit platelets.

The inhibition of selenium-glutathione peroxidase by metal ions was studied by means of a direct spectrophotometric assay that monitors at 237 nm the decrease of GS^? concentration with time. Cadmium (II) and zinc (II) ions were the most potent inhibitors, while silver (I), mercury (II), cobalt (II), and lead (II) inhibited to a lesser extent. Inhibition by these metal ions was competitive with respect to the donor substrate, GSH. Competitive inhibition was verified for cadmium (II) ion by means of an assay employing Ellman's reagent, 5,5′-dithiobis-2-nitrobenzoic acid. Inhibition by cadmium (II) ion was noncompetitive with respect to the acceptor substrate, t-butyl hydroperoxide. Inhibitor constants obtained from Lineweaver-Burk plots and binding constants obtained from Scatchard plots were comparable. Correlation of inhibitor constants with chemical and physical properties showed a dependence on the softness of the metal ion as an acid and also a dependence on ionic size.     

Characterization of histamine release in digitonin-permeabilized rabbit platelets

To manipulate the intracellular milieu of rabbit platelets, permeabilization was performed using digitonin. Permeabilized platelets showed dose-dependent release of histamine, which was stored in granules of rabbit platelets, in response to extracellular calcium ion. As PMA stimulated the release reaction in digitonin-permeabilized platelets, the protein kinase C system, which regulates metabolic processes and cell reactions in intact platelets, was revealed to be working. Cupric phenanthroline also released histamine from permeabilized rabbit platelets dose-dependently, and dithiothreitol inhibited the release strongly. Since cupric phenanthroline is a mild oxidant which catalyzes the formation of disulfide bridges, as in the case of Ca2+-ATPase of sarcoplasmic reticulum, the results suggested that protein cross-linking is implicated in the regulation of the release reaction in permeabilized rabbit platelets.     

Serotonin organelles of rabbit platelets contain synaptophysin

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.     

Morphology of rabbit collecting duct.

Recently the assumed structural and functional homogeneity of the collecting duct (CD) has been questioned. The objective of this study was to determine if heterogeneity occurs in luminal surface membrane structure or in cytoplasmic configuration of cells in the collecting duct or both. Straight segments of cortical and medullary CD were examined in perfusion-fixed rabbit kidneys with scanning electron microscopy (SEM), light (LM) and transmission electron microscopy (TEM). Principal cells were the most abundant cells in all CD regions; intercalated cells comprised 37% of the cell population on the cortex, 18% in the outer medulla, and less than 1% in the inner medulla. SEM revealed two surface patterns among the ciliated principal cells: 1, located in the cortex and outer medulla, with few surface microvilli, and 2, located in the inner medulla, with abundant microvilli. Intercalated cells exhibited four distinctive luminal surface configurations: I, numerous short microvilli; II, both short and elongate microvilli; III, microplicae alone; and IV, both microvilli and microplicae. Intercalated cells with patterns I and II were predominant in the cortex, while cells with patterns III and IV were most common at the corticomedullary junction. TEM confirmed that marked variation existed in cytoplasmic structures of both principal and intercalated cells. These findings may either indicate the presence of several specific types of principal and intercalated cells or reflect different functional states of the principal and intercalated cells. Regardless of their significance, their presence must be considered in studies seeking to establish precise structural-functional relationships in this region of the rabbit renal tubule.     

Multiple forms of monoamine oxidase in rabbit platelets.

Rabbit platelets were found to contain both types A and B MAO activities. The specific enzymatic activity of rabbit platelet MAO was higher for the substrate serotonin than for phenylethylamine. The K_m's for rabbit platelet MAO indicated that the MAO-B enzyme was similar to human platelet MAO and that both MAO-A and MAO-B enzymes in the rabbit platelet are similar to the corresponding forms in the rabbit brain. The drugs clorgyline and deprenyl confirmed the existence of types A and B MAO in the platelet and furthermore indicated that the type A form accounted for approximately 90% of the total enzymatic activity. Amitriptyline at low (micromolar) concentrations selectively inhibited MAO-B activity in both rabbit platelets and brain.     

Production of platelet-activating factor by washed rabbit platelets

Production of platelet-activating factor by washed rabbit platelets under stimulation with the ionophore A23187 was investigated utilizing two groups of platelet preparations. The first platelet preparation contained 0.03 +/- 0.02% contaminating white cells, while the second preparation contained 0.48 +/- 0.27% white cells. The latter preparation produced platelet-activating factor, mainly 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 8.3 +/- 6.3 pmol (mean +/- standard deviation) with a range of 2.6 to 21.4 pmol (n = 9), followed by small quantities of 1-octadecenyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast, there was no production of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by the former platelet preparation having 0.03% leukocytes. These quantitative analyses were carried out by the selected ion monitoring technique and it was concluded that it is necessary to consider the presence of contaminating white cells in studies on the production of platelet-activating factor by platelets.     

Human platelets attenuate oxidant injury in isolated rabbit lungs

Because platelets contain active antioxidant systems, the capacity of platelets to attenuate oxidant lung injury was investigated. Purine and xanthine oxidase were infused into isolated perfused rabbit lungs (IPL) to generate H2O2, thereby causing increased membrane permeability edema. The coinfusion of washed human platelets (1.20 +/- 0.07 x 10(10) cells) attenuated the degree of edema formation as measured by lung weight gain and lung lavage albumin concentration. Electron microscopy of lung preparations demonstrated platelet adherence to capillary endothelial luminal surfaces of oxidant-injured lungs, but there was no evidence of vascular plugging with platelet macroaggregates. The platelet glutathione redox cycle or platelet catalase were inhibited before infusion of platelets into the IPL with purine and xanthine oxidase. Inhibition of the glutathione redox cycle with 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-chloro-2,4-dinitrobenzene, or buthionine sulfoximine prevented platelet attenuation of lung injury. Inactivation of platelet catalase with 3-amino-1,2,4-triazole, however, did not significantly reduce the platelet-induced lung protection. We conclude that the platelet glutathione redox cycle plays a major role in reducing enzymatically generated toxic O2 metabolites and attenuating lung injury.     

Serotonergic agonists stimulate inositol lipid metabolism in rabbit platelets

The metabolism of inositol phospholipids in response to serotonergic agonists was investigated in rabbit platelets. In platelets prelabelled with [3H]-inositol, in a medium containing 10 mM LiCl which blocks the enzyme inositol-1-phosphatase, 5-hydroxytryptamine (5-HT) caused a dose-dependent accumulation of inositol phosphates (IP). This suggests a phospholipase-C-mediated breakdown of phosphoinositides. Ketanserin, a selective 5-HT2 antagonist, was a potent inhibitor of the 5-HT response, with a Ki of 28 nM, indicating that 5-HT is activating receptors of the 5-HT2 type in the platelet. Lysergic acid diethylamide (LSD) and quipazine also caused dose-related increases in inositol phosphate levels, though these were considerably less than those produced by 5-HT. These results show that relatively small changes in phosphoinositide metabolism induced by serotonergic agonists can be investigated in the rabbit platelet, and this cell may therefore be a useful model for the study of some 5-HT receptors.     

Comparative biochemical and ultrastructural studies of P-selectin in rabbit platelets

The role of platelets in thrombotic vascular disease has been widely studied in rabbits. Yet, in rabbit platelets, there is little known about the α-granules, which contain many of the key effector molecules for thrombosis. In this comparative study of rabbit platelets, we have characterized the structure and expression of P-selectin, an α-granule membrane protein that mediates leukocyte adhesion and thrombus propagation. The sequences of tryptic peptides of rabbit P-selectin show an overall sequence identity of 74% with human P-selectin, and 69–77% identity with cow, dog, mouse, rat and sheep P-selectins. The mean (±S.D.) apparent molecular mass of reduced rabbit P-selectin is 117±7 kDa which is 8 kDa larger than the unreduced protein (109±5 kDa). Rabbit P-selectin appears smaller than human P-selectin, but is comparable to other species P-selectins, that have fewer ‘complement regulatory protein’ repeat domains. Cell membrane labeling experiments and antibody binding studies indicate that rabbit P-selectin is nearly absent from the surface of resting platelets (290±30 molecules cell⁻¹). However, cellular activation with thrombin causes nearly a 30-fold increase in expression to 14 200±1100 molecules cell⁻¹. P-selectin is also be expressed on the surface of rabbit platelets activated by other agonists like ADP, A23817 and epinephrine. This selective expression is explained by immunoelectronmicroscopic studies, which show that rabbit P-selectin is sequestered in the intracellular granules of resting platelets. After cell activation by thrombin, P-selectin is found decorating the external membranes of platelet pseudopodia and the surface connected canalicular system. In summary, these studies of P-selectin in rabbit platelets indicate that it is similar in structure, cell localization and expression to human and other species P-selectins. This suggests that studies of P-selectin in thrombosis in rabbits are likely to provide useful insights into the role of this molecule in human thrombotic vascular disease and related conditions.