Altered reabsorption of protein by the renal cortex in rats treated with hypertonic saline or mannitol.

The reabsorption of horseradish peroxidase (HRP) by the proximal tubule cells of rat kidneys was investigated by measuring the concentration of HRP in total particulate fractions of the cortex 1/4 and 1 hr after intravenous injection, and by correlated cytochemical observations. When compared to the corresponding values of the control animals, the concentration of HRP 1 hr after injection was decreased approximately 10-fold in the renal cortex of rats which had received an intravenous injection of hypertonic saline or two subcutaneous injections of mannitol. The plasma clearance and the urinary excretion of HRP were not altered significantly after injection of hypertonic saline, but the plasma clearance was decreased and the urinary excretion increased after injection of mannitol. When the dose of injected HRP was varied, the reabsorption of HRP by the renal cortex was proportional to the dose in the experimental and the control animals. Cytochemical staining for peroxidase activity also showed that the phagosomes and phagolysosomes of the proximal tubule cells contained much less peroxidase in the experimental rats than in the control rats. After injection of mannitol, large vacuoles appeared in the proximal tubule cells. The vacuoles often contained peroxidase-positive granules (phagosomes) which varied in diameter from the limit of microscopic visibility up to several microns. Most of the vacuoles did not react for acid phosphatase activity, but lysosomes were often aggregated around the vacuoles and seemed to release acid phosphatase into the cytoplasm. Certain analogies between the reabsorption of protein and that of water by the proximal tubule cells are discussed.     

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.     

Release of immunoreactive-neuropeptide by rat platelets

Neuropeptide Y, a potent vasoconstrictor and cardiac depressant, is re-leased from sympathetic nerve endings. Its presence in megakaryocytes suggests this peptide might be stored in platelet granules and released during aggregation. Immunoreactive-neuropeptide Y was measured in platelet rich and platelet poor plasma, and was substantially greater in the former. Addition of collagen to platelets resulted in release of neuropeptide Y which paralleled, in a concentration-dependent manner, the degree of platelet aggregation. Adenosine diphosphate, at concentrations which induce only the first phase of aggregation and not the release reaction, caused only a minor release of neuropeptide Y. These results suggest that platelet release could be a major source of circulating neuropeptide Y and could contribute to hemodynamics of pathophysiological states involving platelet activation.     

Does autoregulation of megakaryocytopoiesis occur?

Although a major regulator of thrombocytopoiesis is the number of circulating platelets, several observations suggest that independent alternative regulatory mechanisms may exist. In some situations there is a curious association of megakaryocytopenia and megakarocytic macrocytosis in spite of normal platelet counts. If macrocytosis is considered as a sign of stimulation, this association suggests a cause and effect relationship between decreased numbers and increased size of megakaryocytes. This thesis was tested by examining the delayed effects of sublethal irradiation and the acute effects of hydroxyurea in mice. It was found that megakaryocytopenia and macromegakaryocytosis occurred together and that platelets counts were either normal or only slightly reduced. Therefore it was concluded that normal numbers of platelets could be produced by decreased numbers of megakaryocytes. Megakaryocytopenia appeared to be compensated, in part, by increased size of megakaryocytes, but the mechanism by which this occurred has not been elucidated. It is postulated that a reduction in the number of cells of the megakaryocytic system is sensed by a homeostatic mechanism that then acts to stimulate the cells that are present. This stimulation may then be manifested as macrocytosis of megakaryocytes.     

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.     

RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy and Platelet Generation

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

Thrombin-stimulated effects on megakaryocytopoiesis and pulmonary-platelet interactions

The effects of thrombin stimulation on megakaryocytopoiesis and pulmonary-platelet interactions were investigated before and after administration of the compound to 15 mongrel dogs. Each dog served as its own control. Thrombin was given to encourage the traffic of megakaryocytes into the lung and to study the thrombin-stimulated effects on megakaryocytopoiesis in the bone marrow. Our results showed that thrombin increased the numbers of bone marrow cells in general and megakaryocytes (MK) in particular. In addition, the maturation cycle of megakaryocytes was accelerated and the number of MK migrating into the central venous circulation was nearly doubled. Most of the circulating MK ultimately became sequestered in pulmonary capillaries, where platelets were shed into the arterial circulation. We conclude that thrombin has a major stimulatory effect on megakaryocytopoiesis in the bone marrow and that the lung plays an important role as a vascular filter and regulator of circulating platelet count.     

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin

Summary Using horseradish peroxidase (HRP) as a tracer, we have investigated if the so-called apical tubules (AT) in the kidney proximal tubule cells are directly involved in the endocytic process by carrying the tracer into the cells, or if they are derived from the intracellular membrane compartments. Rat kidney was fixed by vascular perfusion at different time intervals after intravenous injection of HRP and prepared for electron microscopy. An analysis revealed that 0.5 min after injection, invaginations of the plasma membrane and small apical endocytic vesicles, including coated vesicles, were labelled with reaction product, whereas almost all large apical endocytic vacuoles and the AT were negative. The endocytic vacuoles and about 18% of the AT were labelled 1 min after injection. The reaction product in the large endocytic vacuoles was usually seen along the luminal surface of the vacuoles. The AT with reaction product appeared as a branched network, and were frequently connected with the labelled endocytic vacuoles. Three min after injection, reaction product was detected in about 38% of the AT, and thereafter, the percentage increased to about 74% after 7 min. No reaction product was detected in the Golgi complex at any time after HRP-injection. These findings indicate that the AT are probably formed by budding off from the large endocytic vacuoles, rather than being directly involved in the endocytic process.     

The cellular biology of megakaryocytes

Megakaryocytes show a pattern of cellular proliferation and maturation which is unique in mammalian biology. Cells mature to the point of cytoplasmic fragmentation in three major ploidy classes, 8n, 16n, and 32n and the three are fed from a precursor committed stem cell. Two-thirds of the cells belong in the 16n class, and approximately one-sixth in the 8n and 32n classes. The cytoplasm of cells in each ploidy class has a characteristic concentration of granules and demarcation membrane system which appears to be translated into the characteristic features of the platelet progeny from each class. These differ from normal young platelets. Megakaryocytes release fragments of cytoplasm into marrow sinusoids and these differ from platelets in that they do not have the peripheral microtubular bundle or sub-marginal dense tubular system. Transition from fragment to circulating platelet presumably takes place elsewhere in the circulation. With stimulation of platelet production, "stress" platelets are produced, from megakaryocytes which show changes with respect to content of polyribosomes, glycogen and membrane.     

Delivery of lectin-labeled membrane to the trans-Golgi network and secretory granules in cultured atrial myocytes

To examine whether and how internalized plasma membrane components are routed to the compartment of the biosynthetic-exocytic pathway in cultured atrial myocytes, the plasma membrane labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was traced electron microscopically by cytochemical detection of HRP. The WGA-HRP label was internalized via a coated pit-small vesicle pathway and reached vacuoles and endosomes by 3 min. Labeled endosomes comprised vacuoles and tubular elements containing reaction product. By 15 min, similar tubular structures containing reaction product accumulated in the area of the trans-Golgi network (TGN). The labeled TGN consisted of interconnected tubular elements, which often connected to atrial granules containing reaction product. In contrast, neither native HRP nor Lucifer Yellow reached Golgi elements or atrial granules. These results suggest that a proportion of the plasma membrane labeled with WGA-HRP is delivered to endosomes, from which tubules might bud off to transfer the tracer molecules to the TGN, where the lectin conjugate and associated membranes are packaged into atrial granules.     

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.     

Lipid composition of guinea pig platelets and megakaryocytes the megakaryocyte as a probable source of platelet lipids

Platelets are formed by fragmentation of the cytoplasm and plasma membrane of the megakaryocyte in the bone marrow. This study has compared the lipid composition of guinea pig platelets and megakaryocytes. Phospholipids were quantitated by TLC and measurement of lipid phosphorus. Cholesterol and fatty acids were quantitated by GLC. The cholesterol/phospholipid molar ratio was 0.35 in megakaryocytes and 0.55 in platelets. The phospholipid distribution in megakaryocytes was: 9.8% phosphatidylserine, 6.7% phosphatidylinositol, 14.2% sphingomyelin, 40.0% phosphatidylcholine and 29.3% phosphatidy lethanolamine. Platelets contained 11.2% phosphatidylserine, 5.1% phosphatidylinositol, 16.1% sphingomyelin, 38.6% phosphatidylcholine and 29.0% phosphatidylethanolamine. The major megakaryocyte fatty acids were 20.0% palmitic, 16.4% stearic, 20.6% oleic, 13.2% linoleic and 8.2% arachidonic. The major platelet fatty acids were 17.4% palmitic, 17.5% stearic, 11.6% oleic, 12.4% linoleic and 14.6% arachidonic. The minor fatty acids were found in similar proportions in both cells. The major and minor fatty acid compositions of the individual platelet phospholipids reflected those of the megakaryocyte counterparts. The increased arachidonic acid and decreased oleic acid in platelets relative to megakaryocytes were found in all four glycerophospholipids. The similarity of the phospholipid and fatty acid composition of megakaryocytes and platelets suggests that the lipid composition of the platelet is determined by the megakaryocyte.     

Decreased serglycin proteoglycan size is associated with the platelet alpha granule storage defect in Wistar Furth hereditary macrothrombocytopenic rats. Serglycin binding affinity to type I collagen is unaltered

The Wistar Furth (WF) rat has a hereditary defect in platelet formation that resembles gray platelet syndrome of man with a large mean platelet volume and platelet alpha granule deficiency. The alpha granule abnormality is suggestive of a defect in granule packaging and/or stability. Proteoglycans are hypothesized to play a role in granule packaging. Therefore, we have analyzed the structure of the platelet proteoglycan, serglycin, in platelets of WF and normal Wistar rats. Normal and Wistar Furth rats were injected with ³⁵S-sulfate to label platelet proteoglycans via synthesis by the megakaryocytes, and platelets were isolated 3 days later. We found that WF rat platelets have only one-third of the normal proteoglycan mass per unit platelet volume, and the proteoglycans are smaller in hydrodynamic size with shorter glycosaminoglycan chains than those of Wistar rats. However, WF rat platelet proteoglycans showed no defect in binding to collagen on affinity coelectrophoresis gels. We conclude that the structure of WF rat platelet proteoglycans is abnormal, and speculate that this abnormality may contribute to abnormal packaging of the alpha granule contents. Leakage of alpha granule contents into the marrow by platelets and megakaryocytes could perturb the marrow matrix, and promote the development of myelofibrosis noted in gray platelet syndrome. J. Cell. Physiol. 172:87–93, 1997. © 1997 Wiley-Liss, Inc.     

In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk.

Gram-negative bacteria release LPS, which activates Toll-like-receptor-4 (TLR4) in the host, initiating an inflammatory response to infection. Infection increases risk for thrombosis. Platelets contribute to defense from infection and to thrombosis. Experiments were designed to determine whether LPS, through TLR4 signaling, affects platelet phenotype. Platelet responses in wild-type (WT) mice and mice that lack the TLR4 gene (dTLR4) were compared following a single nonlethal injection of LPS (0.2 mg/kg iv). Compared with WT mice, mice without TLR4 had fewer circulating platelets with lower RNA content and were less responsive to thrombin-activated expression of P-selectin but were equally sensitive to aggregation or ATP secretion. One week following the LPS injection, the time it takes for the circulating platelet pool to turnover, the number of circulating platelets, thrombin-induced expression of P-selectin, and collagen-activated aggregation were increased comparably in both groups of mice. Therefore, the change of the platelet pool to an activated phenotype 1 wk after a single exposure to LPS appears to arise from a process that is independent of TLR4. The persistence of the effect 1 wk after the injection suggests that the changes reflect an action of LPS on megakaryocytes and their platelet progeny rather than on circulating platelets, which would have been cleared.     

Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine

Fc receptors on the luminal membranes of intestinal epithelial cells in the neonatal rat mediate the vesicular transfer of functionally intact IgG from the intestinal lumen to the circulation. In addition, there is a low level of nonselective protein uptake, but in this case transfer does not occur. To determine whether a specialized class of endocytic vesicles could account for the selective transfer of IgG, mixtures of IgG conjugated to ferritin (IgG-Ft) and unconjugated horseradish peroxidase (HRP) were injected together into the proximal intestine of 10-d-old rats, and the cellular distribution of these two different tracers was determined by electron microscopy. Virtually all apical endocytic vesicles contained both tracers, indicating simultaneous uptake of both proteins within the same vesicle. However, only IgG-Ft bound to the apical plasma membrane, appeared within coated vesicles at the lateral cell surface, and was released from cells. HRP did not bind to the luminal membrane and was not transferred across cells but was confined to apical lysosomes as identified by acid phosphatase and aryl sulfatase activities. To test the possibility that the binding of IgG to its receptor stimulated endocytosis, HRP was used as a fluid volume tracer, and the amount of HRP taken up by cells in the presence and absence of IgG was measured morphologically and biochemically. The results demonstrate that endocytosis in these cells is constitutive and occurs at the same level in the absence of IgG. The evidence presented indicates that the principal selective mechanism for IgG transfer is the binding of IgG to its receptor during endocytosis. Continued binding to vesicle membranes appears to be required for successful transfer because unbound proteins are removed from the transport pathway before exocytosis. These results favor the proposal that IgG is transferred across cells as an IgG-receptor complex.     

Diminished thrombogenic responses by deletion of the Podocalyxin Gene in mouse megakaryocytes

Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.     

Disorders in the thrombocyte link of hemostasis during endotoxinemia and their correction with the inhibitor of prostaglandin biosynthesis, indomethacin

After 10 minutes of endotoxin Salmonella typhimurium (1 mg/kg) injection into rabbits thrombocytopenia appeared, the aggregation and secretory function of circulating platelets reduced, the transformation of platelet forms from disk-shaped into spheroidal took place. On the surface of plasmatic membranes the pseudopodies and aggregates examining samples of PRP were observed. Indomethacin, blockator of biosynthesis of prostaglandins results in normalisation of morphofunctional properties of platelets.     

Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus.

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.