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An ultrastructural study of ciliated epithelial cells in the ductuli efferentes of young and adult hamsters has revealed that these cells possess dense granules, dense granule clusters, dense bodies and fibrogranular complexes as reservoirs or precursors for ciliogenesis. The dense granules are first seen in the centrosomal region. Later, many dense granules and dense granule clusters appear in the apical portion of the epithelial cells where, subsequently, dense bodies are also found. Finally, the fibrogranular complexes are formed in adults. Morphological evidence strongly suggests that cilia are formed from diplosomal centrioles, de novo centrioles, dense body centrioles, and fibrogranular complex centrioles. Ciliogenesis begins in the fourth day after birth and increases rapidly in the fifth day. After the sixth day, cilia appear to be generated mostly from dense bodies and the total ciliogenesis activities gradually decrease as the animal ages.

Functional and biochemical studies of platelets from human Chediak-Higashi syndrome (CHS) are scarce and/or incomplete. In the present report, the aggregation response to a variety of inducers of platelet aggregation, the content of the dense granule constituents ATP, ADP, serotonin and calcium, the secretion of ATP, ADP, and calcium induced by thrombin, the total content of magnesium, the incorporation of 14C-adenine in the cytoplasmic pool of adenine nucleotides, as well as the content of intracellular cyclic-AMP, have been quantitated in six patients with CHS. Furthermore, data is presented on the kinetics of uptake of radiolabelled serotonin and its storage in human CHS platelets. An abnormal aggregation behaviour was found in all patients. However, the response of CHS platelets to the different inducers studied did not show a uniform pattern. The total content and the maximal amounts of the dense granule constituents secretable by thrombin were greatly decreased in all six patients. Total magnesium content was similar to that of normal platelets. The ATP/ADP ratio was higher than in controls. Uptake of radiolabelled serotonin by CHS platelets closely followed the uptake by normal platelets; during the first 2-3 min, however, incorporation of the amine by CHS platelets came rapidly to a plateau which contrasts with the steady, linear increase in uptake found in controls. CHS platelets loaded with radiolabelled serotonin and gel-filtered, showed a spontaneous release of radioactivity not observed in normal platelets under the same conditions. The cyclic-AMP content of CHS platelets was similar to that of normals. In contrast to platelets from patients with storage pool disease, the secretable calcium from CHS platelets represents a 67% of total platelet calcium (61% in normals), suggesting that the absolute values for the non-secretable portion in CHS platelets must be very low. The results reported confirm the existence of a true storage pool deficiency of the dense granule constituents as a common defect in CHS platelets. The variety of responses among patients, to the different aggregatory stimuli studied, can not be solely ascribed to the storage pool deficiency described.

Summary Cryopreservation of platelet suspensions leads to a severe reduction in the functional responses of these cells. This detect primanly resides in a subpopulation which consists of about half the total platelet mass and shows an increased susceptibility to the Cryopreservation procedure. Following freeze-thawing this subpopulation failed to aggregate upon stimulation with collagen despite a normal appearance of membrane glycoproteins. Also the contents of α granules was similar to the whole suspension and since changes in α and dense granule contents often occur in parallel, the poor aggregability could not be attributed to a defect in secretion granules. In contrast, the generation of phosphatidyl-inositol metabolites, the increase in cytosolic Ca2+ content and the phosphorylation of proteins of mol wt 47 000 and 20 000 seen when normal platelets are activated, were completely absent in the subpopulation. These data reveal a severe defect in stimulus-response coupling mechanisms in about half of the platelets after freeze-thawing. The other half of the suspension endures this treatment relatively unharmed and may be used for transfusion.

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.

Medullary tissue of the normal rat kidney was perfused with 3 percent glutaraldehyde (GA), incubated in 0.5 percent cetyl pyridinium chloride and postfixed in 1 percent OsO4. In comparison with the ordinary fixation with GA and OSO4, the medullary interstitium represented abundant matrical substance that is rich in acid mucopolysaccharides (AMPS) and morphologically represents a diffuse reticular structure consisting of 30 to 150 a thick microfibrils and granular structures of 300 to 500 A in diameter. When chondroitinase was applied before OsO4 treatment, the dense granes disappeared and the microfibrils were replaced by loosely textured 30 A thick microfilaments. After hyaluronidase treatment the microfibrils disappeared and most granules changed into a ring-shaped structure with an electronlucent central portion. These results suggest that the reticular structure consists of microfilaments of hyaluronates and amorphous masking substance of chondroitin sulfates. In the dense granule, hyaluronates become concentrated in the central portion and chondroitin sulfate in the peripheral zone. When perfused with a CPC-containing GA, the medullary interstitium was diffusely filled with a large amount of fine granular substances suggesting the presence of water soluble free AMPS filling the reticular space.

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

Identification and distribution of the precursors of aminestorage organelles in rat megakaryocytes during cell maturation were studied, using the uranaffin reaction for adenine nucleotide. The precursors of the amine-storage organelles appeared as 200–300 nm vesicles having an uranaffin electron dense granule, whereas they appeared as empty vesicles by conventional glutaraldehyde-OsO4 fixation. X-ray probe microanalysis confirmed the existence of U and P in the uranaffin reaction positive vesicles. The precursors appeared in the immature megakaryocytes, especially at the trans(mature) face of the Golgi apparatus, and rapidly increased in number in the maturing cells. The size of the uranaffin granules in the precursory organelles increased gradually during cell maturation and became almost equivalent to the dense body of blood platelets in the final stage of cell maturation.

Serial measurements of platelet volume, platelet content of adenine nucleotides, beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and ex vivo platelet aggregation were made in baboons under basal, steady-state conditions of normal platelet production, and during recovery from acute thrombocytopenia induced by the exposure of flowing blood to spherical glass microbeads. The mean basal platelet count of 509 +/- 107 X 10(9)/l (+/- 1 SD; n = 4) fell acutely to 36.8 +/- 12.6 X 10(9)/l after the insertion of glass bead columns and blood filters, placed distally, for 60 min into surgically implanted arteriovenous-shunts in heparinized baboons. After the irreversible removal of up to 90% of the baseline circulating platelet population, recovery from thrombocytopenia was characterized by a constant rate of increase in circulating platelet counts (115 +/- 11 X 10(9)/l/d) and a rebound thrombocytosis to 1.5 times the basal platelet count after 7 d. Steadystate thrombocytopoiesis was achieved by 3-4 weeks after the onset of thrombocytopenia. Platelet dense granule ADP and ATP decreased significantly from 3.89 +/- 0.20 and 2.33 +/- 0.25 mumol/10(11) platelets respectively at baseline to 2.17 +/- 0.37 and 1.68 +/- 0.37 mumol/10(11) platelets respectively after 7 d (P less than 0.001 in both cases) and normalization was achieved only after 4 weeks. By contrast, the mean platelet volume and platelet content of beta-TG and PF4 did not change significantly throughout the course of study (P greater than 0.1 in both cases). Platelet function, assessed by platelet aggregation ex vivo, demonstrated that platelet function was not impaired despite the significant decrease in dense granule ADP. We conclude that a selective, temporal reduction in platelet dense granule adenine nucleotides reflects changes in the thrombocytopoietic control mechanism secondary to induction of acute thrombocytopenia.

We have developed a model of acute Dacron graft thrombosis in baboons in order to assess platelet alterations secondary to arterial thrombus formation. In this model, thrombus formation was initiated by Dacron vascular grafts inserted as extension segments into chronic arteriovenous Silastic shunts. Following platelet labeling with 111In-oxine, platelet deposition was measured for 1 hour following blood contact under arterial flow conditions using a scintillation camera. Graft platelet activity rapidly increased 40- to 50-fold, plateauing by 1 hour. All grafts produced equivalent reductions in circulating platelet count and blood 111In-platelet radioactivity, demonstrating that the labeled cells were functionally equivalent to the total platelet population. After graft placement, the remaining platelets survived normally. Acute platelet deposition was equivalent on grafts placed 1, 24, 48, and 72 hours following injection of the labeled cells, indicating that a variable delay between platelet labeling and graft imaging was without detectable consequence. Platelet destruction by the graft produced a tenfold increase in plasma levels of platelet factor 4 (PF4) and beta-thromboglobulin (beta TG) but did not modify either the alpha-granule (PF4, beta TG) or dense granule (ADP, ATP) contents of circulating platelets.

When examining peripheral blood, either with phase-contrast illumination or a fixed film and a Romanowsky stain, the smallest elements seen are platelets. These cells usually range from 1 to 3µm in diameter, although an occasional larger cell up to 5µm may be seen. With higher magnification a central area is seen, termed the granulomere because it contains granules, and a peripheral clearer area called the hyalomere. When wet preparations are examined by phase-contrast the granules move within the cell. If a fixed film has been obtained from a finger prick, rather than from venous anticoagulated blood, clumps of platelets are observed due to the triggering of platelet aggregation following the tissue trauma and glass contact. Artifacts may occur in the platelets’ appearance if the blood is collected into anticoagulants and allowed to stand for any length of time. This is particularly so when the anticoagulant used is ethylene diamine tetra-acetic acid (EDTA) in which the platelets swell on incubation. Studies of recalcification of platelet-rich plasma using phase-contrast microscopy showed a rapid change in shape from disc to a spiny sphere and the early appearance of long thin filaments with aggregation into masses of cells which coalesce and lose the granules, and from these radiate strands of fibrin. This phenomenon was called viscous metamorphosis, but examination by electron microscopy shows that each individual cell retains its integrity, and that the changes observed by the light microscope reflect both the release reaction (v. infra) and the redistribution of the cells’ organelles during platelet aggregation as well as changes in the platelets’ shape.

Cells from a variety of animal and human neoplasms can induce the aggregation of platelets in plasma (1–3). While the mechanism of this effect has not been fully elucidated preliminary evidence suggests that several mechanisms may exist. With human tumor cells platelet aggregation may be induced via mechanisms involving the leakage of tumor cell ADP (2, 4, 5; see also Chapters 11 and 12). In rodents aggregation may occur with both whole cells and cell sheddings. This suggests that the aggregating activity of intact cells is mediated by the spontaneous release of some subcellular component. We found that a mixture of membrane vesicles and cytoplasmic dense granules were shed (6). Normal cells are also capable of shedding a small amount of material which is much less active in platelet aggregation (6,7).

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

The neurohemal contact area of the median eminence was examined in adult, young, neonatal, and fetal rats in freeze-fracture preparations. While no specializations of the membranes of axonic terminals abutting on the perivascular space was observed, adjacent areas of the same membranes are rich in signs of hormone release. Signs of exocytosis are defined in the manner of Theodosis et al. (1978). Exocytotic pits with a single dense granule as a core are identified on P-faces along with mounds on corresponding E faces. These features appear near the time of birth, are especially numerous at 3 days, 3 weeks, and 5 weeks, and continue in lesser numbers into adulthood. Even more numerous and appearing even earlier, by the last day of fetal life, were P-face clusters of large particles usually in a pit, and pits without particles. These fall into 2 significantly different categories distinguished by the size of the pit. E face counterparts are large and small protuberances, respectively. Fenestrae of adult size and distribution are observed along the endothelium of portal vessels from the 20th day of fetal life on. Their frequency correlates well with other structural manifestations of a median eminence ready for the onset of functional activity at about the time of birth (Monroe and Paull 1974).

The secretory and aggregation responses of stirred platelets exposed to complement proteins C5b-9 was investigated. C5b-9 assembly on the platelet surface resulted in the release of dense granule adenosine triphosphate (ATP) accompanied by a decrease in sample turbidity, but no detectable cell lysis. Inhibition of cellular protein kinase C completely blocked the C5b-9 initiated release of ATP, confirming the secretory nature of this response. Addition of fibrinogen (up to 1 mg/mL) had no effect on either the release of ATP or the decreased turbidity observed for C5b-9 cells. Addition of the peptides Arg-Gly- Asp-Ser (RGDS) and fibrinogen gamma-chain carboxyl-terminal (gamma 397- 411) at concentrations sufficient to completely block fibrinogen binding to GP IIb-IIIa had no effect on either C5b-9 induced dense granule secretion or the associated turbidity change. Microscopic examination and electronic particle counting of the stirred platelet suspensions confirmed that no aggregation of C5b-9 platelets occurred even when these cells were stirred in the presence of fibrinogen. The capacity of the C5b-9 proteins to initiate platelet secretion without activation of cell surface glycoprotein (GP) IIb-IIIa suggests a mechanism for intravascular dissemination of activated platelets during complement activation in vivo.

Platelet aggregation was measured in 14 patients with systemic lupus erythematosus (SLE) and 13 normal controls. Ten SLE patients (group I) showed decreased responsiveness to collagen, while aggregation was normal in 4 (group II). Group I patients also responded poorly to epinephrine. Platelets from SLE patients did not differ from controls in the production of malondialdehyde in response to N-ethylmaleimide, suggesting intact prostaglandin synthetic pathways. However, a significant decrease (P less than 0.005) in platelet levels of the dense granule constituent, serotonin, was noted in group I SLE patients. Treatment of SLE platelet-rich plasma with deoxyribonuclease (DNase) resulted in enhancement of collagen-induced aggregation in 4 group I SLE patients, but not in 1 group II or 8 normal individuals. These results suggest that defective aggregation in SLE platelets may be partially related to a storage pool deficiency state. However, the ability to restore aggregation to collagen by digestion of platelet-rich plasma with DNase indicates that the defect is reversible and that it is perhaps mediated by plasma or platelet-associated DNA.

Platelets from cattle with the Chediak-Higashi (CH) syndrome are virtually devoid of dense granules, serotonin (5-HT), and stored ATP and ADP. The present study determined how the handling of 5-HT in normal cattle platelets differed from that in CH cattle platelets. Normal and CH platelets accumulated 5-[14C]HT to the same extent. After normal and CH platelets were incubated with 5-HT for 12 h most 5-HT is still intact, indicating that it was protected from metabolism. Part of the newly acquired 5-HT in normal and CH platelets was in a pool that was rapidly released by 5 U/ml of thrombin, suggesting that 5-HT was, in part, within granules. Subcellular fractionation studies showed that, whereas most of the newly acquired 5-HT in normal platelets was located in the dense granule fractions, about one fourth was found in the lighter granule fraction that was enriched in alpha-granules. The dense granule fraction was virtually absent in CH platelets, and most of the granule 5-HT was associated with the lighter granule fraction. The mixed granule fraction from CH platelets accumulated 5-HT but the uptake was about 10% of that from normal platelets. Unlike normal granules the uptake of 5-HT by CH granules was only slightly inhibited by reserpine but was reversed by NH4Cl and nigericin treatment.

The rhoptries of Sporozoan zoites are a set of intracellular organelles supposed to be secretory and to play a role during the invasion of the host cell by these parasites. A prerequisite to the determination of their function is the identification of the nature and properties of their contents. Little data have been obtained so far in this field except in the genus PIasmodium were a 235 kDa protein in P. yoelii (Holder & Freeman 1981) and 2 proteins of 140 and 80 kDa in P. falciparum (Holder, Freeman, Uni & Aikawa 1985; Campbell, Miller, Hudson, Franco & Andriziak 1984) have been identified in the rhoptries of erythrocytic merozoites by monoclonal antibodies (McAb). Schwartzman (1986) has obtained McAbs specific for the rhoptries of Toxoplasma gondii tachyzoites but has not identified the corresponding antigens. We report here on the McAbs characterization of a 42 kDa protein located in the rhoptries of tachyzoites and bradyzoites of T. gondii.

Addition of NaF to washed platelets produces a dose-dependent and transient elevation of the intracellular free calcium concentration ([Ca++]i), thromboxane B2 (TxB2) generation and dense granule release, all of which are significantly inhibited when the extracellular calcium concentration ([Ca++]e) is reduced with EGTA. Inhibition of platelet cyclo-oxygenase by acetylsalicylic acid (ASA) does not affect NaF-induced elevation of [Ca++]i and dense granule release in the presence of 1 mM [Ca++]e. Pre-incubation of the platelets with the phorbol ester TPA produces a marked inhibition of NaF-induced elevation of [Ca++]i and TxB2 generation without affecting dense granule release. Thus, NaF may have more than one site of action. Pretreatment of the platelets with the selective protein kinase C inhibitor H7 prevents TPA induced inhibition of NaF mediated rise in [Ca++]i and TxB2 generation. Thus we propose that NaF induced calcium mobilisation is analogous to receptor-operated calcium mobilisation in platelets, as it is readily inhibited by protein kinase C activation or by the reduction of [Ca++]e and is independent of platelet cyclo-oxygenase activity.

Removal of extracellular sodium decreased clacium mobilization from intracellyular stores induced by thrombin in aspirin-treated human platelets. ATP and serotonin secretion were also significantly reduced. Secretion was positively correlated with calcium mobilization, but the presence or absence of sodium did not modify the slope of the regression line. Half-maximal secretion was reached when [Ca2+]1 was increased by about 0.1 μM. Calcium mobilization induced by the divalent cation ionophore ionomycin was not modified by sodium removal. Secretion induced by ionomycin was much smaller than the thrombin-induced one for the same increases of [Ca2+]1. These results suggest that the presence of external sodium is required for normal thrombin-induced calcium release from the intracellular stores and hence for dense granule secretion. However, secretion cannot be only attributed to the increase of cell [Ca2+]1 but also to other process(es) which are not affected by external sodium.

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.

The role of platelet prostanoids, ADP and 5HT in initial attachment, spreading and aggregation of platelets on collagen substrates (CI, CIII, CIV, CV, CC) was studied. A positive linear correlation was found between thrombi-like aggregate formation on collagen substrates and production of platelet prostanoids. No correlation was established between platelet aggregation and 14C-5HT release. Thrombi-like aggregate formation was completely inhibited by indomethacin and TXA2/PGH2 antagonists (13-APA and BM 13.177). Both 13-APA and BM 13.177 had no effect on platelet spreading, while indomethacin inhibited this process by 25%. The ADP-scavenger system (CP/CPK) inhibited platelet aggregation and spreading by 25-30%. Initial attachment was not influenced by aspirin, indomethacin and CP/CPK. The data obtained indicate that platelet aggregation on collagen substrates is mediated by PGH2 and TXA2 production. These compounds slightly affect the platelet spreading. Both platelet spreading and aggregation on collagen substrates are only partially mediated by ADP and 5HT release. Initial attachment of platelets does not depend on the release reaction and PGH2/TXA2 synthesis.

Polymerized type I calf skin collagen induced a time-dependent specific binding of 125I-fibrinogen to washed human platelets. Binding occurred more rapidly in a shaken rather than in an unstirred system. It was linear in the range 0.05-0.3 microM added fibrinogen and was saturated at higher fibrinogen concentrations (more than 0.8 microM). Scatchard analysis showed a single population of binding sites (16530 +/- 5410 per platelet) with a Kd = 0.53 +/- 0.23 microM. Collagen-induced 125I-fibrinogen binding to platelets was completely inhibited by ADP antagonists such as creatine phosphate/creatine phosphokinase and AMP, and partially inhibited by pretreatment of the platelets with aspirin. With both normal and aspirin-treated platelets a close correlation was observed between the amount of 125I-fibrinogen bound and the extent of dense granule secretion. Our results confirm that fibrinogen becomes bound to platelet surface receptors during collagen-induced platelet aggregation and suggest that secreted ADP is an essential cofactor in this process.

The cerebrospinal fluid (CSF)-contacting neuron is not a paraneuron. The reasons why a chapter of the present volume is devoted to this neuron are: (1) It has certain cell-biological features in common with paraneurons (Nakai et al. 1977a, b); (2) it is homologous to retinal and pineal paraneurons, as has already been discussed (see pp. 241, 248); and (3) it has recently been regarded as the prototype of neurons, possibly sharing chemoreceptive and secretory features with bipolar paraneurons (see below).

The ultrastructure of human T and B leukemic cells is compared in this review. Both cells types can display smooth and villous surfaces, and nuclear pockets. B-cells generally contain more abundant rough endoplasmic reticulum (RER) than T-cells. On the other hand, pre-B-cells have as little RER as T-cells. Clustered dense granules, which exhibit lysosomal enzyme activities, seem to be a reliable ultrastructural marker of T-cells. Accumulation of glycogen particles is seen in T- but not in B-cells. Endoplasmic reticulum- or ribosome-related abnormalities, such as intracisternal accumulation of immunoglobulin, annulate lamellae, and a ribosome-latnella complex, are sometimes found in B-cells.

The distribution of two platelet antiheparin proteins, platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) measured by radioimmunoassays and of antiheparin activity measured by a chromogenic assay, was determined in human platelet granule fractions separated by sucrose density centrifugation. Subcellular fractions were characterized by the levels of serotonin (dense granule marker) and β-N-acetylglucosaminidase (lysosomal enzyme marker). PF4 and LA-PF4 antigens and antiheparin activity showed an identical distribution pattern with the highest specific activities occurring in a fraction lighter than that containing serotonin. The fraction with the highest specific activity of β-N-acetylglucosaminidase was observed at a lighter sucrose density than the fraction most enriched in platelet antiheparin proteins. These results indicate that PF4, LA-PF4 and heparin-neutralizing activity are located in the same granule fraction (α-granules) which is different from both the serotonin and the β-N-acetylglucosaminidase-containing fractions. The PF4 and LA-PF4 amounted to 13% of the total proteins of α-granules as determined by radioimmunoassay and SDS polyacrylamide gel densitometric scanning.

Summary. This study was designed to evaluate the usefulness of platelet density analysis in the detection of acquired storage pool defects in human patients. Two groups of patients were investigated: 19 subjects affected with a myeloproliferative disorder (group I) where abnormal platelets are released from the megakaryocytes and 11 patients hospitalized in an intensive care unit (Group II) where normal platelets are injured in the circulation. Platelet density distribution after isopycnic centrifugation on a discontinuous stractan density gradient, dense granule markers (serotonin, ATP and ADP) and alpha granule markers (intraplatelet beta-thromboglobulin and platelet factor 4) were simultaneously determined. An increased proportion of the percentage of light platelets was observed in 16 patients of group I and nine of group II; an increased ATP/ADP ratio was observed in 12 patients of group I and 10 of group II. Both the tests were abnormal in 11 patients of group I and nine of group II. In group I, the level of serotonin was low and was related to the percentage of light platelets. The alpha granule specific proteins were normal in the two groups. These results indicate that platelet density analysis may serve as a screening test to detect exhausted platelets in human diseases.

Some aspects of spermiogenesis have been studied in the testis of the teiid lizard Cnemidophorus lemniscatus lemniscatus by electron microscopy. Shortly after the acrosomal vesicle is lodged in a nuclear concavity of the spermatid, a dense granule differentiates in the center of the subacrosomal space. It is cone-shaped and shows a longitudinal striation. Its base applies to the acrosomal membrane and, through this, to the acrosomal granule. Its rounded vertex causes a depression of the nuclear membranes which, initially juxtaposed, separates at this point to form a vesicle. The granule develops and becomes a rod when spermiogenesis is advanced and the subacrosomal space has taken the form of a secondary cap. The rod is cylindrical, retains its original striation and has a convex acrosomal end. It encloses the vesicle formed by the nuclear envelope in its base and follows the apex of the nucleus. Meanwhile, the acrosomal granule loses its identity and the acrosomal cap is filled with a dense substance, in which a fringe of translucent material differentiates. This fringe lies in the dorsal and apical margins of the acrosome and is incompletely divided by longitudinal crests of the dense acrosomal substance. A projection of the Sertoli cell forms an accessory cap which envelops the acrosome and is in turn covered by the cytoplasm of the spermatid, constituting an intricate association. Two reflex membranes underlie the plasmalemma in the outer surface of the projection of the Sertoli cell. They are continuous with one another at their ends and with the cell membrane in the edge of pores. In the peripheral cytoplasm of the spermatid facing the accessory cap, numerous microtubules run longitudinally. By means of thin membranes some are interconnected or connected with the plasmalemma, from which they seem to originate.

Platelet aggregation and secretion of granular contents require metabolic energy. This implies the existence of a control mechanism that adjusts the rate of energy producing pathways to the energy need of these functions. Such a control function has been attributed to the level of metabolic ATP, to the adenylate energy charge (AEC =(ATP + 1/2ADP)/(ATP + ADP + AMP) and to the velocity of energy generation and consumption (ATP-turnover). In this study we investigated which factor dominates in control of dense granule (3H-serotonin)-, α-granule (β thromboglobulin)- and lysosomal granule (N acetyl β glucosaminidase) secretion. Human gel- filtered platelets were incubated in glucose-free, CN containing medium. Under these conditions ATP-generation only took place in glycogenolysis, which alone was unable to maintain ATP homeostasis. Consequently, metabolic ATP and AEC fell. Addition of glucose restored glycolytic ATP resynthesis wich restored the AEC but the loss of ATP was for the main part irreversible due to hypoxanthin formation. With this system a broad range of metabolic ATP levels and AEC’s could be obtained both at decreasing ATP turnover (before glucose addition) and at increasing ATP turnover (after glucose addition). Analysis of secretion velocity of dense-, α- and lysosomal granules after initiation with thrombin (5 U/ml) showed that at decreasing turnover the secretion velocity of all types of granules depended on both metabolic ATP level and AEC. In contrast, at increasing ATP turnover the correlation between secretion velocity and metabolic ATP level or AEC was lost. Instead, dense granule secretion was faster and lysosomal granule secretion was slower than expected on the basis of metabolic ATP level or AEC, whereas α-granule secretion showed intermediate levels. The data indicate that the three types of granule secretion differ in their dependence on metabolic energy and that apart from metabolic ATP and AEC, ATP turnover is of crucial importance for secretion of granular contents.

The purpose of this study was to isolate distinct populations of canine neutrophil granules and to compare them with neutrophil granules from other species. Size, shape, density, and content of canine neutrophil granules were determined. Neutrophils obtained by Ficoll-Hypaque sedimentation were homogenized, and granule populations were separated by isopycnic centrifugation on a linear sucrose gradient (rho, 1.14 to 1.22 g/ml). The most dense granule population (rho, 1.197 g/ml) contained all of the myeloperoxidase, beta-glucuronidase, and elastase, more than half of the acid beta-glycerophosphatase, and most of the lysozyme. The population with intermediate density (rho, 1.179 g/ml) contained lactoferrin, vitamin B12-binding protein, and the remainder of the acid beta-glycerophosphatase and lysozyme. The least dense granule population did not contain a major peak of any of the enzymes or binding proteins tested but was distinguished by density and morphology. The size and shape of the granules were determined from scanning electron micrographs and assessment of shape was aided by transmission electron micrographs. By these methods three populations of canine neutrophil granules were characterized and named: myeloperoxidase granules, vitamin B12-binding protein granules, and low-density granules.

Human chorionic gonadotropin (HCG) was localized in human placenta at the ultrastructural level by an immunocytochemical method which linked the antigen HCG to a peroxidase label through the antigen-antibody reactions of an immunoglobulin-peroxidase bridge. Material on the maternal surface of the apical plasma membrane and in the cisternae of rough endoplasmic reticulum of the syncytiotrophoblast displayed strong immunostaining for HCG and the outer surface of the basal plasma membrane appeared equivocally immunoreactive. Lamellae and vesicles of the Golgi complex of the syncytium lacked electron-opaque reaction product indicative of immunostaining for HCG. Deposits of peroxidase reaction product in the large variety of cytoplasmic dense granule varied from negligible to abundant in immunostained and in control specimens alike and, accordingly, the large granules appeared to contain intrinsic peroxidase but not HCG. The question of the immunoreactivity of the small variety of cytoplasmic dense granule was not conclusively settled, but the occurrence of reactive and unreactive small granules in both immunostained and control specimens weighed against the presence of HCG in these rather infrequent granules. The cytotrophoblast lacked evidence of immunoreactivity.

The pancreatic islet cells of the toads (Bufo vulgaris formosus) captured in December was observed by the electron microscope.1. Pancreatic islet cells of the toad can be classified into the A-, B-, third, fourth and fifth types. The A- and B-cells are the most common, whereas the third and the fifth type cells are less common and the fourth type cells are the least common.2. The α-granules of the A-cells of the toad agree in ultrastructural properties with those of other animal species of the vertebrate. Each of them consists of a spherical granule core of high density and a smooth limiting membrane applied closely to it; the electron lucent space between the both is in general narrow. A smaller number of α-granules with less dense core are also found. It is one of the characteristics of the A-cells that they contain a considerable amount of glycogen granules dispersed in the whole cytoplasm.3. The β-granules of the B-cells of the toad consist of several, irregularly arranged, rod-shaped and crystalloid cores of low density and of the limiting membrane loosely surrounding them. The presence of filament bundles in the cytoplasm represents a characteristic of the B-cells. In the B-cells elongated mitochondria with unique appearance occur sometimes singly or in small number in which longitudinal cristae mitochondriales are densely packed in a lamellar structure. Huge elongated crystalloid bodies encountered exclusively in the B-cells may correspond to an enlarged and modified type of those special mitochondria.4. The third and fourth type islet cells are regarded as the variants of the A-cells, since they possess specific granules similar to α-granules and contain in cytoplasm variable amounts of glycogen granules. The specific granules of the third type cells are remarkably small and the limiting membrane is attached closely to the dense granule core, so that the light space between membrane and core is usually obscure. Specific granules with a less dense core are frequently observed. In this islet cell type the specific granules are dispersed only sporadically and the mitochondria are generally smaller than in other islet cell types. In the fourth type cells the numerous specific granules coincide in their size and ultrastructural properties with α-granules, although their cores are generally less dense and show coarser texture; in addition frequent elliptic granules are characteristic.5. The fifth type islet cells are characterized by the poorly developed Golgi apparatus, by the mitochondria with indistinct mitochondrial membrane and by the numerous free ribosomes in spite of the absence of the cisterns of endoplasmic reticulum. The cores of the specific granules are in general moderately lucent showing coarse texture and the limiting membranes are closely applied to the cores, but specific granules with a dense core may also be encountered. The fifth type islet cells seem to correspond to the D-cells described in previous electron microscopic observations on the pancreatic islets in other animals.6. The third and fifth type islet cells are each gathered in small cell groups which, juxtaposed to each other, are located between the A- and the B-cell groups in the islet. This location coincide with that of the silber-positive or argyrophil islet cells (A1-cells of HELLMAN and his collaborators) observed in the light microscopy. It may be therefore possible that the third and fifth type islet cells, both or one of them, correspond to the silber-positive cells.7. The specific granules of the islet cells were confirmed to be formed in the Golgi complex, but the extrusion mechanism of them could not been clarified in the present study. However, the following finding seems to be worthy of attention: The limiting membranes of certain specific granules frequently become discontinuous and sometimes even disappear, so that the granule cores can be discharged

Publisher Summary This chapter discusses the differentiation of macrophages. The mononuclear phagocytes that comprise the reticuloendothelial system (RES) represent a large, morphologically heterogeneous group of cells that are widely distributed throughout all organs and tissues. These cells may be most simply divided into an intravascular, immature component characterized by the blood monocyte and a more mature tissue counterpart, the macrophage or histiocyte. Both the formation of acid phosphatase-positive, phase-dense granules and the cell content of acid hydrolases could be blocked through the use of inhibitors of protein synthesis. The macrophage contains two types of lysosomes. The first and more obvious is the dense granule. This, by definition, is a secondary lysosome or digestive body, which contains both endogenously synthesized hydrolases and substrates taken up by endocytic activity. The primary lysosome of the macrophage would then be the Golgi vesicle in which enzyme is packaged and transported to the pinocytotic vacuole. Although the secondary lysosome is already involved in the digestive process, it may share its contents with a membrane-bounded structure derived from a subsequent endocytic event.

Sympathetic nerve endings of the mouse pineal were examined by qualitative and semiquantitative electron microscopy at various times of the day under usual lighting conditions (12 hours of light and 12 hours of darkness per day). The nerve endings contained a mixed population of both small granular vesicles and small agranular vesicles. The small granular vesicles, 650 A in mean diameter, were characteristic of adrenergic nerves. They were subdivided, on the basis of the size of the granules, into two types, Types 1 and 2. The Type 1 granular vesicles contained a dense granule which, though varying in size, did not completely fill the vesicle and was often located eccentrically within the vesicle. The Type 2 granular vesicles had a large granule that almost filled the vesicle. The small agranular vesicles were also subdivided, according to their shape, into two types, Types 1 and 2. The Type 1 agranular vesicles were spherical with a mean diameter of 650 A, and the Type 2 agranular vesicles, elliptical or flat, measuring 800×300 A in mean long and short diameters. The proportions of vesicles of the above types, except for the Type 1 agranular vesicles, varied diurnally in correlation with the cycles of day and night. During the light period of the day, both the granular and agranular vesicles were almost constant in proportion. After the onset of darkness, however, all the vesicles except for Type 1 agranular vesicles underwent remarkable changes in the proportion of population. The proportion of Type 1 granular vesicles decreased remarkably until midnight, but at the end of the dark period it was increased again. Simultaneously with a decrease in proportion, the granules appeared to decrease in size. After the onset of the light period the Type 1 granular vesicles increased in proportion. The Type 2 granular vesicles, though less numerous in number, also showed the same tendency in proportion as the Type 1 granular vesicles, and they were almost absent at midnight. The small agranular spherical vesicles of Type 1 remained almost unchanged in proportion throughout the light and dark periods. The Type 2 agranular flat vesicles increased after the onset of darkness. At the end of the dark period they were again reduced in proportion. In addition to the four types of small vesicles, occasional large granular vesicles having a diameter of more than 1000 A were also contained within the nerve endings. They remained unchanged in both proportion and nature throughout the day.

Elongated, dumbbell-shaped mitochondria, containing longitudinal stacks of lamellae, have been described in the liver of rats subjected to certain toxic agents, such as ammonium carbonaie or dinitrophenol; their occurrence and particular morphology have been considered to be the expression of degenerative processes, and the intramitochondrial lamellae likened to myelin figures [1, 2]. Dumbbell-shaped or elongated forms of mitochondria, with apparently concentric longitudinal cristae have been observed in rats following administration of thyroxine in excess [3]. Lehninger has pointed out that the latter extended or dumbbell-shaped configurations may be related to mitochondrial growth and division [4].

A specimen from a patient with an ectopic pinealoma located in the third ventricle was investigated by light and electron microscopy.

Paired segmental nerve bundles in Periplaneta americana which join the lateral cardiac nerve cords near each ostial value in the abdominal heart are composed of at least three ordinary axons each enclosed by layers of glial tissue. The majority of the bundle cross section is composed of a large number of neurosecretory axons characterized by the absence of glial folding and which can be grouped into three separate types by small electron dense, large electron dense, or electron opaque axoplasmic granules. Axons with small dense granules make neuromuscular junctions with the heart and synapse near motor nerves in the lateral cardiac cord. The formation of release sites in the lateral cord by both small and large electron dense granule axons from the segmental nerve is confirmed. The electron opaque granule in some axons of the segmental nerve is assumed to be precursor material. A fourth distinct axon containing a large electron transparent. granule of unknown origin was found in the lateral cardiac nerve cord and makes a neuromuscular junction with the heart. Also found in the lateral cardiac cord is a neuron cell which is not a cardiac ganglion cell with cytological evidence of neurosecretory synthesis. Neurosecretory material delivered from the ventral ganglion chain to the vicinity of the heart is considered to comprise in part a cardio-regulatory system in the cockroach with some analogy to other invertebrates. No other physiological role is known for the extensively developed cardiac neurosecretory system, although the multitude of apparent active processes suggests participation other than simple control of the heart.

Corpora cardiaca of freshly molted male Leucophaea maderae and Periplaneta americana, prepared in the standard manner for electron microscopy, are seen to possess numerous membrane-limited “sacs” of granules. These “sacs”, reaching 5 μ in width, appear to be bulbous axon terminals of brain neuro-secretory cells or processes of intrinsic glandular cells. The terminals are often in close juxtaposition with corpus cardiacum cells. Closely packed granules within some of these structures approach 150 mμ and in others approach 300 mμ in diameter. The granules are frequently seen to have a limiting membrane. Although the nuclei and cell bodies of corpus cardiacum cells have been seen rather infrequently, the micrographs included reveal that these cells possess elements of rough-surfaced endoplasmic reticulum, mitochondria and, occasionally, a large, dense granule. Tissue elements are sometimes separated by unlined, optically empty spaces, by tracheoles, or by extensions of the connective tissue sheath, which frequently penetrate between tissue elements to a considerable depth.

During spermatogenesis in Gerris remigis, chromatoid bodies appear in the spermatocytes and persist to the-mid-spermatid stage. These structures consist of numerous, parallel tubules, which measure approximately 500 A in diameter. The tubules are arranged in hexagonal array, and contain dense granules that resemble ribosomes. The chromatoid body may be secretory in function, or may be involved in intracellular transport.

In the course of electron microscopic investigations of the fundus mucosa of the mouse stomach, a few cells of an unknown type were found by chance in the deep portions of the glands. These cells are characterized by two different kinds of specific granules in their cytoplasm, one of which being large and less dense, and the other one being small and dense. The large less dense granules resemble zymogen granules of the chief cell, which are formed by the rough endoplasmic reticulum and Golgi system. The small dense granules are quite similar to the secretory granules of the “basal granulated cell,” and are considered to be formed in the Golgi complex. Release figures of the small dense granule were not observed, numerous granules, however, were observed in close contact with the basal cell membrane. The occurrence of these two kinds of granules in one cell suggests that the basal granulated cell and the zymogenic cell originate from the same entodermal stem element.

In mice exposed to whole body X-irradiation, the proximal tubular cells show marked cytoplasmic alterations after 48 hours. The cytoplasm contains hypertrophied smooth endoplasmic reticulum that appears as tubular profiles which forms an interconnecting lattice that courses throughout large areas of the cytoplasm. In unilaterally nephrectomized animals and animals exposed to X-irradiation 4 hours following unilateral nephrectomy, numerous whorl-like membranous structures occur in the basal and medial cytoplasm of proximal tubular cells after 48 hours. It is suggested that these membranous structures represent a substantial absolute increase in new membranes for these cells. Many dense lamellated granules composed of parallel alternating dense and light bands that are arranged in concentric arrays are observed in the apical and medial zones of the proximal tubular cells. The internal lamellar pattern of the dense granule is similar to the myelin figures obtained from phospholipid water systems reported by several investigators. After 3 months, the prominent cytoplasmic alterations are not apparent; however, numerous vacuoles containing large accumulations of lipid, along with many dense granules and filaments, appear throughout the apical and basal cytoplasm of the proximal tubular cells.

This report deals with the events following phagocytosis of foreign material in Kupffer cells of rat livers. It is a study of early stages of phagocytosis of ferritin and Latex particles. All observations were made between 1 and 10 minutes after intravenous injection.

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers’ platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers’ platelets, presumably due to a biogenesis defect. Additionally, 25–29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7–9%) and autophagosome-like structures (0–3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.

Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelet. Although several platelet SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8(-/-) platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8(-/-) mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis.

Apicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite’s actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement. In this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites. We demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.

Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplaninfl/fl mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.

Background: Platelet function disorders are a common cause of a bleeding problem. While many rare and severe forms of platelet function disorders are well studied, many common platelet function defects are uncharacterized. In a family with uncharacterized platelet secretion defects, we identified a single base pair insertion in the gene RUNX1 by exome sequencing. We report on the clinical and laboratory phenotype of the defect in this family. Methods: Bleeding histories of affected and unaffected family members, obtained using a standardized questionnaire, were scored using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT). Laboratory data evaluated included blood counts, platelet aggregation responses, dense granule ATP release findings (lumiaggregometry) and platelet dense granule counts, evauated by whole mount preparations and electron microscopy. Affection status was based on the recorded opinion of the subject's hematologist, which concurred with diagnostic laboratory findings. Exome sequencing was performed on the index case, followed by evaluation of all family members for the candidate mutation by polymerase chain reaction (PCR) amplification and Sanger sequencing. Results: Family members investigated (median age: 25.5, range 1-69] included 5 males (affected: n=4, unaffected: n=1) and 3 females (affected: n=2, unaffected: n=1). ISTH BAT bleeding scores for affected members were elevated (median: 10.5, range 4-20), unlike unaffected family members (n=2) and healthy controls (n=40) (ranges: 0-1 and 0-2). Platelet counts were low in 2/6 affected individuals (109 platelets/L : affecteds: median: 164, range: 125-169). While unaffected family members had unremarkable platelet function findings, the affected individuals had absent or reduced dense granule secretion with all agonists tested (including thrombin, collagen, epinephrine, U46619 and arachidonic acid), and in aggregation tests, they had absent secondary aggregation with epinephrine, reduced maximal aggregation with collagen (1.25 and 5.0 μg/mL) and thromboxane analogue U46619, variable responses to arachidonic acid (reduced in 4/5 affecteds), and normal responses to ADP and ristocetin. Only some (3/6) affected family members had dense granule deficiency (lower reference interval limit: 4.9/platelet; affecteds: median: 5.2, range: 4-6). A single base pair insertion (A) in exon 6 of the RUNX1 gene (NM_001754.4:c.583dup) was identified in the index case using exome sequencing. The mutation results in a reading frame shift at codon Ile195, that is expected to end in a premature termination codon 17 positions downstream. Sanger sequencing further confirmed this mutation was present in 6/6 affected family members and it was absent in both unaffected family members. Further evaluation of the family history indicated that only one family member (deceased aunt of the index case) had developed leukemia or myelodysplasia although her mutation status is unknown. Conclusion: We identified a novel, frameshift RUNX1 mutation in a family with an inherited platelet function disorder associated with increased bleeding symptoms. This variant has not been previously reported in exome sequencing of over 60 000 individuals as documented by the ExAC consortium. The variable platelet count and dense granule numbers among affected individuals in this family emphasize the importance of phenotyping multiple family members. RUNX1 defects should be considered as a potential cause of inherited abnormalities in dense granule secretion, even in individuals without dense granule deficiency or a striking family history of leukemia/myelodysplasia. Disclosures No relevant conflicts of interest to declare.

Reduced levels of the neurotransmitter serotonin in blood platelets is a clinical symptom characteristic of individuals with Down's syndrome. To investigate the possible involvement of the Cu/Zn-superoxide dismutase (CuZnSOD) gene, which resides at the Down locus on chromosome no. 21, in the etiology of that symptom, we examined blood platelets of transgenic mice harboring the human CuZnSOD gene. It was found that platelets of transgenic CuZnSOD animals, which overexpress the transgene, contain lower levels of serotonin than nontransgenic littermate mice, due to a reduced rate of uptake of the neurotransmitter by the dense granules of the platelets. We found that the pH gradient (delta pH) across the dense granule membrane, which is the main driving force for serotonin transport, was diminished in dense granules of transgenic-CuZnSOD. Furthermore, a significantly lower than normal serotonin accumulation rate was also detected in dense granules isolated from blood platelets of Down's syndrome individuals. These findings suggest that CuZnSOD gene dosage is affecting the dense granule transport system and is thereby involved in the depressed level of blood serotonin found in patients born with Down's syndrome.