Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia

Apoptotic hepatocytes have been demonstrated to represent an important signal for transmigration of leukocytes sequestered in sinusoids during endotoxemia in vivo. Beside leukocytes, platelets and their adhesion to endothelial cells and leukocytes have been implicated in inflammatory liver injury. Using in vivo multifluorescence microscopy, we examined the possibility that hepatocellular apoptosis causes both leukocytes and platelets to colocalize within the sinusoidal microvasculature of endotoxemic livers. We further addressed the issue whether cellular colocalization with apoptotic hepatocytes is cause or consequence of apoptosis. Intraperitoneal exposure of rats with LPS (5 mg/kg) induced liver injury after 6 and 16 h, as given by nutritive perfusion failure (20 +/- 2 and 21 +/- 2%), intrahepatic leukocyte (60 +/- 10 and 121 +/- 48 cells/mm(2)), and platelet (12 +/- 4 and 34 +/- 4 cells/mm(2)) accumulation as well as parenchymal cell apoptosis (4 +/- 1 and 11 +/- 2 cells/mm(2)) and caspase cleavage (4.7 +/- 2.4- and 7.0 +/- 3.0-fold increase; P < 0.05 vs. saline-exposed controls). Higher doses of LPS (10 mg/kg ip) further increased intrahepatic leukocyte and platelet accumulation but not the extent of parenchymal apoptosis. Detailed spatial analysis revealed colocalization of leukocytes (range 12-24%) but barely of platelets (<6%) with apoptotic hepatocytes in all endotoxemic groups studied. It is of interest, however, that platelets were found at increasing rates in colocalization with leukocytes at 6 and 16 h after LPS exposure (5 mg/kg LPS: 7 +/- 3 and 25 +/- 6%; 10 mg/kg LPS: 11 +/- 4 and 14 +/- 1%). Platelet-leukocyte events significantly correlated with the extent of caspase cleavage as an indicator of tissue apoptosis (P < 0.05; r = 0.82). Blockade of apoptosis by a pan-caspase inhibitor caused a significant reduction of leukocyte adherence and platelet-leukocyte colocalization on LPS exposure. On the other hand, leukocytopenic animals revealed reduced hepatocyte apoptosis, although values still exceeded those of controls, and in leuko- and thrombocytopenic animals, hepatocyte apoptosis was found reduced to control values. Taken together, LPS-associated hepatocyte apoptosis seems to be initiated by circulating blood cells that become adherent within the liver but might also contribute to further sustain the inflammatory cell-cell response.     

Involvement of Kupffer cells in lipopolysaccharide-induced rapid accumulation of platelets in the liver and the ensuing anaphylaxis-like shock in mice

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here).     

In Vitro and In Vivo Characterization of Ultraviolet Light C-Irradiated Human Platelets in a 2 Event Mouse Model of Transfusion

UV-based pathogen reduction technologies have been developed in recent years to inactivate pathogens and contaminating leukocytes in platelet transfusion products in order to prevent transfusion-transmitted infections and alloimmunization. UVC-based technology differs from UVA or UVB-based technologies in that it uses a specific wavelength at 254 nm without the addition of any photosensitizers. Previously, it was reported that UVC irradiation induces platelet aggregation and activation. To understand if UVC-induced changes of platelet quality correlate with potential adverse events when these platelets are transfused into animals, we used a 2-event SCID mouse model in which the predisposing event was LPS treatment and the second event was infusion of UVC-irradiated platelets. We analyzed lung platelet accumulation, protein content in bronchoalveolar lavage fluid as an indication of lung injury, and macrophage inflammatory protein-2 (MIP-2) release in mice received UVC-irradiated or untreated control platelets. Our results showed UVC-irradiated platelets accumulated in lungs of the mice in a dose-dependent manner. High-doses of UVC-irradiated platelets were sequestered in the lungs to a similar level as we previously reported for UVB-irradiated platelets. Unlike UVB-platelets, UVC-platelets did not lead to lung injury or induce MIP-2 release. This could potentially be explained by our observation that although UVC treatment activated platelet surface αIIbβ3, it failed to activate platelet cells. It also suggests lung platelet accumulation and subsequent lung damage are due to different and separate mechanisms which require further investigation.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Platelet activation may explain the storage lesion in platelet concentrates

While the exact nature of the dysfunction of stored platelets is not known, it is generally agreed that the platelet's metabolic activity with lactate accumulation presents a significant impediment to prolonged storage. There is an increasing body of evidence that stored platelets have become activated in the preparation and handling of platelet concentrates. Changes in platelet function and structure in concentrates can be explained in terms of sequelae of activation, especially heightened metabolic activity and activation-specific changes in surface glycoproteins on stored platelets. With the use of inhibitors of platelet activation in the preparation of platelet concentrates, the loss of platelet function and integrity is less rapid and platelet metabolic rate is decreased during an extended storage period. Surface levels of glycoprotein Ib, normally decreased during prolonged storage of platelets, are well-preserved in the presence of activation inhibitors. When the use of inhibitors is combined with replacement of plasma with an artificial medium, platelets stored for up to 20 days appear to be metabolically and structurally intact and responsive to stimuli. In summary, platelet activation appears to play a major role in the generation of the storage lesion in platelet concentrates.     

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia.

A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snap-frozen. During infusion of echistatin (2.3 micrograms/kg/min), circulating platelet counts decreased from 331,000/mm3 to 167,000/mm3. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate 111Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.     

Wortmannin对凝血酶诱导的人血小板聚集和磷脂酰肌醇三磷酸累积的影响

 Ｗ ｏｒｔｍ ａｎｎｉｎ 是肌醇磷脂 ３ 激酶的不可逆抑制剂．用比浊法分析血小板聚集;肌醇磷脂用３２ Ｐ 磷酸钠标记,用氯仿和甲醇抽提,用 Ｔ Ｌ Ｃ和放射自显影分析,研究了 Ｗ ｏｒｔｍ ａｎｎｉｎ 对凝血酶诱导的人血小板聚集和磷脂酰肌醇三磷酸（ Ｐ Ｉ Ｐ３）累积的影响．结果显示, Ｗ ｏｒｔｍ ａｎｎｉｎ 对凝血酶（５００ Ｕ／ Ｌ）诱导的人血小板聚集有抑制作用,这种抑制作用在一定范围内呈剂量依赖关系（２０～８０μｍ ｏｌ／ Ｌ）．凝血酶（５００ Ｕ／ Ｌ）诱导人血小板 Ｐ Ｉ Ｐ３ 的累积, Ｗ ｏｒｔｍ ａｎｎｉｎ 对此累积有抑制作用,这种抑制作用在一定范围内呈剂量依赖关系（４０～１６０ μｍ ｏｌ／ Ｌ）．结果提示： Ｗ ｏｒｔｍ ａｎｎｉｎ 可能是潜在的抗血小板药物,抑制凝血酶诱导的人血小板聚集主要与其抑制 Ｐ Ｉ Ｐ３ 的累积有关．结果也提示,肌醇磷脂 ３ 激酶在血小板活化中起重要作用．     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.     

Murine platelets are not regulated by O-linked beta-N-acetylglucosamine

It is generally appreciated that platelets derived from diabetic patients display increased responsiveness to low levels of agonists. O-GlcNAcylation has been linked to hyperglycemia-related effects in other tissues; therefore we examined this modification in platelets to determine if O-GlcNAcylation affects platelet function. This post-translational modification consists of an N-acetylglucosamine attached to serine and/or threonine residues. We examined O-GlcNAc levels in platelets from a hyperglycemic murine model of Type I diabetes with known hypersensitivity to agonists and a Type II diabetes model (ob/ob) lacking detectable alterations in the aggregation profile. Neither model showed marked increases in protein O-GlcNAcylation. Treatment of platelets with multiple O-GlcNAcase inhibitors led to O-GlcNAc accumulation on multiple platelet proteins. However, the inhibitor-induced accumulation of this modification does not correlate with any gross alterations in platelet aggregation. These data suggest that while the modification occurs in platelets, their activity is not globally sensitive to O-GlcNAc levels.     

Passive and active mechanisms trap activated CD8+ T cells in the liver

The liver is a site where activated CD8(+) T cells are trapped and destroyed at the end of an immune response. The intrahepatic accumulation of activated murine TCR transgenic CD8(+) T cells was significantly reduced when either ICAM-1 or VCAM-1 was blocked by specific Ab. These two adhesion mechanisms account for essentially all the trapping of activated CD8(+) T cells in the mouse liver. Although the ICAM-1-mediated trapping depends on the capacity of the vasculature and/or the parenchymal cells to present Ag, the accumulation of cells through VCAM-1 does not require Ag recognition. Thus, Ags expressed by the non-bone marrow-derived cells in the liver actively cause CD8(+) T cell accumulation through TCR-activated ICAM-1 adhesion, but the liver can also passively sequester activated CD8(+) T cells that do not recognize intrahepatic Ag, through VCAM-1 adhesion.     

Impaired cAMP metabolism associated with abnormal function of thrombopathic canine platelets

The effects of forskolin and 1-Methyl-3-isobutyl-xanthine on thrombopathic platelet function and cyclic AMP accumulation were examined. The concentration of forskolin required to inhibit gamma-thrombin- induced platelet aggregation and secretion by 50% was significantly lower for thrombopathic than for normal platelets. This inhibition was accompanied by a marked elevation of cyclic AMP. 1-Methyl-3-isobutyl-xanthine, alone or in combination with forskolin, augmented both the cyclic AMP accumulation and the inhibition of platelet function. These results demonstrate that cyclic AMP metabolism is abnormal in thrombopathic platelets and imply that cyclic AMP-phosphodiesterase activity is impaired.     

Stereoselective accumulation of the beta-receptor blocking drug atenolol by human platelets.

We have studied the stereochemistry of accumulation of the hydrophilic beta-adrenoceptor antagonist rac-atenolol by human platelets in vitro. The accumulation was slow, not reaching equilibrium until 90 min at 37 degrees C. The uptake was temperature dependent with the accumulation at 37 degrees C being 3-4 times greater than at 4 degrees C. The accumulation was also stereoselective at 37 degrees C, favoring the active (-)-enantiomer over the (+)-enantiomer by 2.3-fold. Reserpine, but not desipramine, inhibited the platelet accumulation of rac-atenolol enantiospecifically. This uptake profile is different from the platelet uptake of lipophilic beta-blockers, which is characterized by nonspecific membrane binding, but similar to the carrier-mediated accumulation of the neurotransmitter norepinephrine by storage granules within the platelet.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Antigen presentation by liver cells controls intrahepatic T cell trapping, whereas bone marrow-derived cells preferentially promote intrahepatic T cell apoptosis.

Systemic activation and proliferation of CD8(+) T cells result in T cell accumulation in the liver, associated with T cell apoptosis and liver injury. However, the role of Ag and APC in such accumulation is not clear. Bone marrow chimeras were constructed to allow Ag presentation in all tissues or alternatively to restrict presentation to either bone marrow-derived or non-bone marrow-derived cells. OVA-specific CD8(+) T cells were introduced by adoptive transfer and then activated using peptide, which resulted in clonal expansion followed by deletion. Ag presentation by liver non-bone marrow-derived cells was responsible for most of the accumulation of activated CD8(+) T cells. In contrast, Ag presentation by bone marrow-derived cells resulted in less accumulation of T cells in the liver, but a higher frequency of apoptotic cells within the intrahepatic T cell population. In unmodified TCR-transgenic mice, Ag-induced T cell deletion and intrahepatic accumulation of CD8(+) T cells result in hepatocyte damage, with the release of aminotransaminases. Our experiments show that such liver injury may occur in the absence of Ag presentation by the hepatocytes themselves, arguing for an indirect mechanism of liver damage.     

Accumulation of platelets at sites of antigen-antibody-mediated injury: a possible role for IgE antibody and mast cells.

Circulating 51Cr-labeled platelets accumulate at skin sites in which a reversed passive Arthus reaction has been induced. The accumulation is biphasic in time and is accompanied by an increased vascular permeability. Increased permeability itself, however, will not produce localization of platelets. A similar platelet accumulation was observed upon injection of compound 48/80 or anti-IgE antibody into the skin and this was not altered in rabbits depleted of complement or neutrophils. Activation of skin mast cells and release of a platelet-activating factor (PAF) is suggested as a mechanism for the effect produced by anti-IgE and compound 48/80. The first phase of platelet accumulation in the Arthus reaction was also unaffected in rabbits depleted of neutrophils or complement, which may suggest a role for IgE antibody and mast cells. The second phase of accumulation was diminished in complement-depleted animals and abrogated in rabbits without neutrophils, suggesting a complement and neutrophil-mediated process but which still might be mediated through mast cell activation by neutrophil cationic protein.     

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia

Summary A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snapfrozen. During infusion of echistatin (2.3 µg/kg/min), circulating platelet counts decreased from 331000/mm³ to 167000/mm³. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate ¹¹¹Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.     

Carnitine inhibits arachidonic acid turnover,platelet function,and oxidative stress

Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.     

The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na⁺-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15 mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H⁺-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.