Ultrastructural morphometric observations on serial sectioned human blood platelet subpopulations

The relationships between the volume of human platelets and their cytoplasmic organelles were studied by morphometric analysis. Platelets were separated into four density-dependent subpopulations on an arabino-galactan gradient. In vitro activation of platelets was effectively prevented by maintaining them at a constant ambient temperature of 37 degrees C. Serial sections were cut through platelets, morphometrically analyzed and the platelets reconstructed. The volumes of the individual platelets and their constituent granules, mitochondria and open canalicular systems (OCS) were calculated. Individual organelles were counted. The mean volumes of the platelets of the subpopulations decreased significantly as density decreased (p = 0.01). Also, as the density of platelets decreased, there was a decrease in the mean unit volume of their granules (p = 0.003). In contrast, independent of platelet volume or density, the OCS occupies about 10% of the platelet volume. These findings indicate that it is possible to prevent in vitro platelet activation by maintaining their environment at 37 degrees C. Our study confirms the direct relationships between platelet volume and density; and platelet density and granule content. There is no ready explanation for the constant relationship between platelet volume and that of the OCS.     

Rearrangement of the open-canalicular system of the human blood platelet after incorporation of surface-bound ligands

Summary The three-dimensional configuration of the membrane system in human blood platelets following administration of cationized ferritin (CF) was reconstructed by means of thick serial sectioning in combination with high-voltage electron microscopy (HVEM).Surface-bound CF was rapidly internalized at 37 ° C by the platelets, and a conglomerate of vacuolar and tubular structures containing CF was formed in their interior. Three-dimensional reconstruction of these incorporated membranous structures revealed that most of the internalized structures were interconnected with each other and that the number of sites at which they opened onto the cell surface was significantly reduced compared with the number found in intact platelets. In some cases, the openings completely disappeared. This may indicate that most of the internalized membranous structures originate from the open canalicular system (OCS), and that, following the incorporation of the ligand, the OCS gradually loses its connections with the surface membrane forming a conglomerate of the ligand-containing membranous structures in the interior of the platelet.     

The surface-connected canalicular system in the sinus endothelial cells of rat spleen

The existence of a surface-connected canalicular system in the splenic sinus endothelial cells of the rat has been demonstrated by transmission electron microscopy with lanthanum nitrate acting as a tracer for the extracellular space. In addition, the three-dimensional arrangement of the canaliculi has been revealed by computer-aided reconstruction. The surface-connected canalicular system of the sinus endothelial cells consists of slender canaliculi that are branched, anastomosed, and that show continuity with the plasma membrane. They twist in and out among the organelles and are often found in close apposition to the spherical invaginations of the plasma membrane and run alongside them. Canaliculi which are not infiltrated by lanthanum nitrate take the form of electron-lucent tubules and are accompanied by numerous spherical invaginations of the plasma membrane. From a computer-aided reconstruction, the canaliculi, which invaginate from various sites of the plasma membrane, have been found to be continuous with each other and to penetrate to the surface of the sinus endothelial cell; they also branch and anastomose to form a complex network in the cytoplasm. Although the surface-connected canalicular system in blood platelets and thrombocytes is believed to function as the main route for the discharge of granules and the uptake of foreign materials and also to take part in the storage and transport of calcium, it is unclear at present whether the network of the surface-connected canalicular system in splenic sinus endothelial cells has any physiological significance.     

Cellular distribution of CD63 antigen in platelets and in three megakaryocytic cell lines

Summary CD63 is a 53 kDa lysosomal membrane glycoprotein that has been identified as a platelet activation molecule. We investigated the localization of CD63 antigen in platelets and in three megakaryocytic cell lines (K562, HEL and CMK11-5) using flow cytometry and immunoelectron microscopy. Flow cytometry showed that a monoclonal antibody directed against CD63 bound to 8.1% of unstimulated platelets and 59.2% of thrombin-stimulated platelets. Immunoelectron microscopy demonstrated that CD63 antigen was distributed randomly inside unstimulated platelets, while it was localized in the open canalicular system of washed platelets and on the cell membranes of thrombin-stimulated platelets. Flow cytometry detected CD63 on 16.4% of HEL cells, 31.2% of K562 cells, and 43.2% of CMK11-5 cells. Immunoelectron microscopy demonstrated that CD63 was localized in the granules and on the surface membranes of HEL cells, in the vesicles and on the membranes of K562 cells, and in the granules and vesicles as well as on the membranes of CMK11-5 cells. Thus, the distribution of CD63 differed markedly among these three megakaryocytic cell lines.     

Ultrastructure of buffalo,Syncerus caffer,platelets: Comparison with bovine and human platelets

Platelets are found in the blood of all mammals and serve the same basic hemostatic functions in all. Species differences in the morphology and activities of platelets in human and domesticated animals have been observed, but there is little published information regarding the blood pictures of wild animals. In this study, the ultrastructure of buffalo platelets was compared with that of bovine and human platelets. Buffalo platelets were found to be smaller than human platelets and intracellularly had larger α-granules, possessed distinctive dense granules and a more distinct microtubuli system, but they lacked the open canalicular system observed in the human type. This morphology is similar to that of cattle platelets. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of factor VIII/von Willebrand factor antigen in human platelets

Specific antibodies against anti-human FVIII/vW protein were isolated by affinity chromatography on glutaraldehyde-activated gel (Ultrogel AcA22). They were coupled directly with peroxidase or visualized with anti-rabbit IgG (sheep)-peroxidase (Institut Pasteur). Fab fragments of the same specific antibodies were prepared to enhance the intracellular penetration and coupled to peroxidase. In washed human platelets, staining was observed on the plasma membrane and in the canalicular system, whereas in previous studies whole specific antibodies incubated with fixed platelets showed the labeling only on the plasma membrane. After thrombin activation, the release of granules containing FVIII/vW protein was better visualized in the surface canalicular system. This localization was discussed in regard to the exocytosis process: membrane fusion, granule labeling.     

Immunocytochemical localization of factor VIII/von Willebrand factor antigen in human platelets

Specific antibodies against anti-human FVIII/vW protein were isolated by affinity chromatography on glutaraldehyde-activated gel (Ultrogel AcA22). They were coupled directly with peroxidase or visualized with anti-rabbit IgG (sheep)-peroxidase (Institut Pasteur). Fab fragments of the same specific antibodies were prepared to enhance the intracellular penetration and coupled to peroxidase. In washed human platelets, staining was observed on the plasma membrane and in the canalicular system, whereas in previous studies whole specific antibodies incubated with fixed platelets showed the labeling only on the plasma membrane. After thrombin activation, the release of granules containing FVIII/vW protein was better visualized in the surface canalicular system. This localization was discussed in regard to the exocytosis process: membrane fusion, granule labeling.     

Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy

Blood platelets from 10 normal human subjects have been examined with a sensitive differential interference contrast (DIC) microscope. The entire transformation process during adhesion to glass is clearly visible and has been recorded cinematographically, including the disk to sphere change of shape, the formation of sessile protuberances, the extension and retraction of pseudopodia, and the spreading, ruffling, and occasional regression of the hyalomere. The exocytosis of intact dense bodies can be observed either by DIC microscopy, or by epifluorescence microscopy in platelets stained with mepacrine. Details of fluorescent flashes indicate that the dense bodies usually release their contents extracellularly, may do so intracytoplasmically under the influence of strong, short wavelength light on some preparations of mepacrine-stained platelets. The release of one or more dense bodies leaves a crater of variable size on the upper surface of the granulomere. Such craters represent the surface component of the open canalicular system and their formation and disappearance can be directly observed. Because these techniques permit quantitation of several parameters of motility which are not readily observable by other techniques, it is suggested that high extinction DIC microscope examination may become a rapid and useful method of studying congenital and acquired platelet disorders. Many features of platelet transformation have been confirmed and extended by scanning electron micrographs. These can in turn be interpreted by reference to time-lapse films of living platelets.     

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.     

Redistribution of alpha-granules and their contents in thrombin- stimulated platelets

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.     

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Changes in the proportion of A- and B-types of granules containing atrial and brain natriuretic peptides in atrial myocytes in vasorenal hypertension in rats

The system of cardiac natriuretic peptides (NP) is a very important factor opposing the effects of the rennin-angiotensin-aldosterone system (RAAS), sympathoadrenal system and vasopressin to reduce blood pressure. In the present study, we analyzed the release of atrial and brain natriuretic peptides in the myocytes of the right atrium in rats by the quantitative morphometric method using double immunocytochemical labeling of atrial granules comprising NP in vasorenal hypertension induced by the renal artery ligation. The decrease in the total amount of secretory granules in atrium myocytes by 18% was detected on the 30th day after the operation. The number of the A-type granules was reduced by 53%, and the number of B-type granules was increased by 64% in comparison with the intact animals. Our data indicate activation of NP secretion in vasorenal hypertension. The results suggest that high arterial pressure might be explained by reduced expression of NP receptors in this pathology.     

Discrepancy of protein kinase C translocation and platelet aggregation--immunocytobiochemical evidence

We searched for possible relationships between platelet aggregation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) or thrombin and the translocation of protein kinase C. Using monoclonal antibodies against subspecies of protein kinase C, we noted a predominant expression of the isozyme, type II, in human platelets (M. Watanabe, M. Hagiwara, K. Onoda, and H. Hidaka, 1988, Biochem. Biophys. Res. Commun. 152, 642). Analysis of the subcellular distribution of protein kinase C revealed that 65% of the kinase activity was present in the cytosolic fraction in unstimulated platelets, with the remaining activity in the membrane fraction. Treatment of platelets with 100 nM TPA resulted in a greater than 60% decrease in protein kinase C activity in the cytosolic fraction and a greater than 200% increase in the activity in the membrane fraction, within 10 min after treatment. Translocation of the enzyme was also found after treatment of platelets with thrombin, although the response was of lower magnitude than that induced by TPA. Similar results were obtained by immunoblotting using MC-2a, an anti-type II protein kinase C monoclonal antibody. We also examined localization of the enzyme, by electron microscopic immunocytochemistry. The presence of type II protein kinase C seemed to be localized mostly in hyaluromeres and not in granulomeres. When platelets were fixed just after the addition of TPA (within 1 min), protein kinase C was localized at the submembranal region with no remarkable change in shape but there was a decrease in the number of granules in the cytoplasma and the open canalicular system was dilated. We then investigated the effects of cytochalasin B, W-7, ML-9, and H-7 on TPA-induced platelet aggregation and the translocation of protein kinase C. W-7 and ML-9 potently inhibited platelet aggregation but none of these compounds hampered the translocation. Thus, activation of protein kinase C may not be a complete requirement for the initiation of platelet aggregation.     

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostacyclin on ultrastructural features of human platelets in vitro

Examining platelets plasma-membrane by freeze-etching (FE)-technique irregularly placed among the “particles”, some larger formations, called by us “protuberances” can be seen. These protuberances may represent the stomata of the open canalicular system (OCS) and the morphological aspect of an exocytotic process. Addition of prostacyclin to platelet rich plasma (PRP) leads to a decrease of the average value of protuberances which is statistically significant in comparison to control platelets.     

Membrane bilayer balance and platelet shape: morphological and biochemical responses to amphipathic compounds

Activated platelets adopt a characteristic spiculate morphology. A wide variety of anionic and zwitterionic amphipathic compounds were found to effect a similar shape change and to cause the open canalicular system to become less prominent. Several cationic amphipaths reversed thrombin-, PAF-, and amphipath-induced spiculation and restored the discoid shape. Higher concentrations of cationic amphipaths caused the cells to assume spheroid and indented forms, and caused the canalicular system to appear more prominent. Three amphipaths were studied further to address possible mechanisms underlying their morphological effects. Dilauroylphosphatidylcholine was found to induce spiculation without causing the changes in protein phosphorylation and inositide metabolism generally associated with platelet activation. Two other amphipaths, chlorpromazine (which induced sphering) and dilauroylphosphatidylserine (which caused spiculation followed by sphering) caused specific changes in protein and/or lipid phosphorylation, which may be responsible for some, but not all, of the morphological effects of these compounds. To account for these findings, we propose that platelet shape can be influenced by changes in the plasma membrane bilayer balance. Agents that bind to the membrane outer monolayer are accommodated by spiculation; those that bind to the inner monolayer are accommodated by sphering.     

Cytochemical localization of ouabain-sensitive (K+)-dependent p-nitrophenyl phosphatase (transport ATPase) in human blood platelets

The ultrastructural cytochemical localization of a potassium-dependent oubain-sensitive nitrophenyl phosphatase (transport ATPase) activity in human blood platelets is described. This potassium-dependent nitrophenyl phosphatase activity was not affected by 5 mM levamisole, indicating that the reaction product identified was not due to nonspecific alkaline phosphatase activity. The K+-dependent nitrophenyl phosphatase was strictly localized to the platelet plasma membrane, while the open canalicular system and dense tubular system were devoid of reaction product. In contrast, (Ca2+,Mg2+)-activated ATPase activity was predominantly localized in the open canalicular system and dense tubular system with very little cytochemical activity expressed at the plasma membrane. These data demonstrate a relative segregation of these enzymes into unique membrane compartments of the human platelet. Such data may be useful with regard to identification of purified membrane fractions from platelets and may be significant with regard to the understanding of the function(s) of the different membrane compartments of the human platelet.     

Blood platelets of the cheetah (Acinonyx jubatus)

The blood platelet count and platelet morphology of 22 adult cheetahs was investigated. The platelet counts of the animals displayed a normal distribution, with a mean count of 344×10⁹/l and a mean platelet volume of 11 fl. Morphological and ultrastructural features of the cheetah platelets revealed the typical platelet morphology of anuclear cells, with granules scattered throughout the cytoplasm. The characteristic surface canalicular system and microtubules were present. True cross‐sections of the platelets had a mean area of 2.146 µm², circumference of 6.805 µm, and mean minimum and maximum projections of 1.000 µm and 2.933 µm, respectively. Zoo Biol 23:263–271, 2004. © 2004 Wiley‐Liss, Inc.     

Platelet membrane responses to surface and suspension activation

Exposure of blood platelets to foreign surfaces or to potent agonists in suspension results in dramatic changes in physical appearance and conversion from a nonsticky state. The transformation to the sticky state is associated with exposure of the fibrinogen receptor, GPIIB-IIIa, which is hidden in resting, discoid platelets. Recent studies employing fibrinogen coupled to gold (Fgn-Au) as an electron-dense probe have suggested that GPIIb-IIIa receptors not only become exposed in surface-activated platelets, but undergo a reorganization not observed in suspension-activated cells. Discoid platelets do not bind Fgn-Au; however, the bodies and extended pseudopods of dendritic forms are covered with Fgn-Au particles. Conversion of dendritic platelets to spread forms is accompanied by movement of receptor-ligand complexes away from peripheral margins into a concentrated mass in cell centers over the inner filamentous zone of the cytoplasm. Movement of the Fgn-Au particles to cell centers during spreading was considered due to the transmembrane action of the newly assembled actin filaments. We have carried out similar experiments on surface- and suspension-activated platelets with Fgn-Au and latex particles. GPIIb-IIIa receptors move Fgn-Au particles on outer membranes of surface- and suspension-activated platelets to channels of the open canalicular system. Treatment with cytochalasin B prevents assembly of actin filaments in surface- and suspension-activated platelets, and dissociates residual actin from the cell membranes, circumferential microtubules and organelles with which they interact. However, cytochalasin B does not prevent removal of Fgn-Au to channels of the open canalicular system. Thus, reorganization of fibrinogen receptors on surface- and suspension-activated platelets is due to the particles, and not to the fibrinogen, although fibrinogen is required to link gold to the receptor. The surface membrane has its own detergent and cytochalasin B-resistant cytoskeleton for directed transport of ligand-receptor complexes, independent of the internal assembly and contraction of actin into an inner filamentous zone.