Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-β-structure of ...misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN
-denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.
Abstract Background and aims Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component ...sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. Methods Mitochondrial or endoplasmic reticulum-dependent apoptosis was induced by exposure of macrophages to etoposide or thapsigargin/fukoidan, respectively. Results Cell death induced by these compounds was inhibited by S1P as inferred from reduced annexin V binding, TUNEL staining, and caspase 3, 9 and 12 activities. S1P induced expression of the inhibitor of apoptosis protein (IAP) family proteins cIAP1, cIAP2 and survivin, but only the inhibitor of survivin expression YM155 and not the cIAP1/2 blocker GDC0152 reversed the inhibitory effect of S1P on apoptosis. Moreover, S1P activated signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) and the stimulatory effect of S1P on survivin expression and inhibitory effects on apoptosis were attenuated by STAT3 or JAK2 inhibitors, S3I-201 or AG490, respectively. The effects of S1P on STAT3 activation, survivin expression and macrophage apoptosis were emulated by HDL, HDL lipids, and apolipoprotein (apo) M-containing HDL, but not by apoA-I or HDL deprived of S1P or apoM. In addition, JTE013 and CAY10444, S1P receptor 2 and 3 antagonists, respectively, compromised the S1P and HDL capacities to stimulate STAT3 activation and survivin expression, and to inhibit apoptosis. Conclusions HDL-associated S1P inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression. The suppression of macrophage apoptosis may represent a novel mechanism utilized by HDL to exert its anti-atherogenic effects.
It is known that valproate inhibits platelet functions; however, the exact mechanisms are not clearly identified. We studied 12 healthy adult volunteers (1 female, 11 male; age range 31.7 ± 7.8 ...years) before and after valproate 500 mg and compared the results to levetiracetam 1000 mg as a control substance and placebo. The study had a crossover and double-blind design. A blood sample was taken before and 90 min after medication intake, because the times to maximum serum concentration (T
) are 1.5 h for levetiracetam and 1 to 3 h for valproate. We analysed changes in platelet, erythrocyte, and leukocyte cell count and in platelet functions (CD62 expression (P selectin), thrombin binding, and fibrinogen binding). We found no significant differences in all cell counts before and after different study drugs. After valproate intake, but not after placebo or levetiracetam intake, the fibrinogen binding significantly decreased and the CD62 expression significantly increased resulting in decreased platelet aggregation. Our data suggest that the platelet dysfunctions reported for valproate result from decreased fibrinogen binding and from increased CD62 expression. This phenomenon might be one reason for the increased bleeding risk under valproate and cannot be observed for levetiracetam.
Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association ...with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.
Summary
1. Prospective and interventional studies demonstrate an inverse relationship between plasma high‐density lipoprotein (HDL)–cholesterol and the incidence of coronary artery disease. Although ...the atheroprotective effects of HDL are usually attributed to the reverse cholesterol transport, in which HDL shuttles cholesterol from cells in the arterial wall to the liver, other mechanisms are also under investigation.
2. Platelets are involved in both the initiation and progression of atherosclerotic lesions. In addition, the formation of thrombi over ruptured atherosclerotic plaques results in the narrowing or complete occlusion of coronary arteries. Current experimental evidence suggests that HDL may exert antiplatelet effects and thereby counteract the development of atherothrombotic vascular disease.
3. In vitro studies show that HDL inhibits agonist‐stimulated platelet aggregation, fibrinogen binding, granule secretion and liberation of thromboxane A2. Inhibitory effects of HDL are mediated, in part, by scavenger receptor type B1 and/or the apolipoprotein E receptor apoER2/LRP8 and are linked to the induction of intracellular signalling cascades encompassing stimulation of protein kinase C, cytoplasmatic alkalization and generation of nitric oxide.
4. Populational studies demonstrate that there is an inverse association between plasma HDL levels and recurrent venous thromboembolism. In addition, HDL–cholesterol has been identified as an independent predictor of acute platelet thrombus formation. The administration of reconstituted HDL particles in humans attenuates ex vivo platelet activation.
5. The present review summarizes recent advances in understanding HDL–platelet interactions and discusses the potential use of HDL‐like particles in the therapy of thrombosis.
As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl ...immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Platelets perform many functions in hemostasis but also in other areas of physiology and pathology. Therefore, it is obvious that many different function tests have been developed, each one conceived ...and standardized for a special purpose. This review will summarize the different fields in which platelet function testing is currently in use; diagnostics of patients with bleeding disorders, monitoring patients' response to anti-platelet therapy, monitoring in transfusion medicine (blood donors, platelet concentrates, and after transfusion), and monitoring in perioperative medicine to predict bleeding tendency. The second part of the review outlines different methods for platelet function testing, spanning bleeding time, and platelet counting as well as determining platelet adhesion, platelet secretion, platelet aggregation, platelet morphology, platelet signal transduction, platelet procoagulant activity, platelet apoptosis, platelet proteomics, and molecular biology.
OBJECTIVE—Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P ...receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P.
APPROACH AND RESULTS—Low-density lipoprotein receptor–deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4, CD8) and activated (CD69/CD8, CD69/CD4) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon-γ production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203–treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-γ–induced protein-10 production; IκB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor κB and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-γ–stimulated bone marrow macrophages, respectively.
CONCLUSIONS—Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.
Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary ...monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies
in
vitro is limited. Immortal proliferating cell lines, such as the human THP-1 monocytic leukemia cell line, are therefore often used instead of primary cells. Transfection is a useful tool to study the function of gene products, but transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages with subsequent differentiation into mature THP-1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. In this study, we describe a simple and rapid approach for efficient transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages using a modified Nucleofection-based approach. The protocol maintains cell viability and functionality, thus allowing efficient transfection of THP-1 cells combined with subsequent differentiation of transfected THP-1 cells into mature macrophages.
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