| 1. |
- Bergstrand, Jan, et al.
(författare)
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Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine-diphosphate and thromboxaneA2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.
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| 2. |
- Bergstrand, Jan, et al.
(författare)
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Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
- 2019
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Ingår i: Nanoscale. - : ROYAL SOC CHEMISTRY. - 2040-3364 .- 2040-3372. ; 11:20, s. 10023-10033
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Tidskriftsartikel (refereegranskat)abstract
- Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.
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| 3. |
- Gustafsson, Amanda Jabin, et al.
(författare)
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ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor
- 2011
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Ingår i: Molecular and Cellular Endocrinology. - : Elsevier BV. - 0303-7207 .- 1872-8057. ; 333:1, s. 8-19
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca2+ concentration ([Ca2+](i)) remains unknown. We measured [Ca2+](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary beta-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca2+](i) in the form of a peak followed by a plateau dependent on extracellular Ca2+. NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca2+](i). The ADPr-induced [Ca2+](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP3 receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPrinduced [Ca2+](i) increase. ADPr increased [Ca2+](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors.
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| 4. |
- Li, Nailin
(författare)
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Platelet and leukocyte activation and their interaction : in experimental prothrombotic and inflammatory states
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Atherosclerotic disease is associated with inflammation and thrombosis, both of which involve multi-cellular activation and interaction. The present work has investigated the mechanisms of platelet leukocyte cross-talk in vitro, and explored the possibility of multi-cellular activation in vivo in prothrombotic and inflammatory models in healthy volunteers. Whole blood flow cytometric methods for measuring platelet-leukocyte aggregates (PLAs) were developed and evaluated. The methods involved minimal sample manipulation, avoiding centrifugation or red cell lysis, and no or only mild post-incubation fixation, to minimize in vitro artefacts. Antibodies that block ligand-receptor interactions had little effect in unstimulated samples but totally inhibited agonist-induced PLA formation, indicating that the assays reflect circulating PLAs closely. Gating on leukocyte fluorescence improved assay efficiency. Data from these studies show that all leukocyte subtypes can form heterotypic conjugates, and that activation of either platelets or leukocytes can enhance PLA formation. Granulocytes and monocytes predominate among PLAs. Multi-ligand-receptor systems are involved in PLA formation, but P-selectin bridging to PSGL-1/CD15s is the principal bridging mechanism under most conditions. Activation of leukocytes with fMLP evoked platelet activation. The effect did not depend on PLA formation, and was markedly inhibited by PAF antagonism or 5-lipoxygenase inhibition. Collagenactivated platelets evoked leukocyte activation. The effect was not inhibited by a thromboxane A2 receptor antagonist, which abolished collagen-induced platelet P-selectin expression, but was inhibited by blocking PLA formation, indicating that the effect depended on cell-cell contact. Interestingly, GPIIb/IIIa blockade by an nonpeptide inhibitor, SR121566, inhibited platelet-leukocyte cross-talk, and a cocktail containing antibodies to P-selectin, GPIIb/IIIa, and CD 1 1b/CD 18 abolished fMLP-induced leukocyte activation. Thus, platelet-leukocyte cross-talk involved multiple mediators and mechanisms, and adhesion molecules seem to be important in cellular signaling during platelet and leukocyte activation. Strenuous exercise provoked a prothrombotic state, and endotoxin injection induced systemic inflammation in humans. Both interventions resulted in multicellular activation in vivo and enhanced thrombin generation. Platelet activation was shown by increased circulating P-selectin positive platelets and platelet-platelet aggregates, elevated plasma soluble P-selectin levels, and enhanced platelet responsiveness to in vitro stimulation. Leukocyte activation was shown, with increased CD11b expression in circulating leukocytes, elevated plasma elastase levels, as well as enhanced leukocyte reactivity to in vitro stimulation. This resulted in enhanced platelet-leukocyte interaction, as evidenced by increased circulating PLAs and enhanced agonist-induced PLA formation in vitro. Endothelial activation and thrombin generation were shown by markedly increased plasma vWF and F1+2 levels, respectively. Aspirin ingestion at a daily dose of 500 mg reduced platelet P-selectin expression at rest slightly, and inhibited neutrophil responsiveness to fMLP stimulation, but did not counteract the prothrombotic effects of exercise. Adenosine infusion at a dose of 40 [my]g/Kg/min induced leukocytosis in the absence of endotoxemia, probably due to inhibition of leukocyte adhesion and migration on/into the vessel wall. Adenosine infusion attenuated endotoxemia-induced platelet and leukocyte activation moderately, as shown by modest inhibition on platelet reactivity and more marked inhibition of leukocyte adhesion and migration. The latter also resulted in the retention of more activated leukocytes and PLAs in the circulation, and might thus limit leukocyte-mediated tissue damage. Taken together, the present studies suggest that thrombosis and inflammation are closely related pathophysiological processes that involve multicellular activation of platelets, leukocytes, and endothelial cells and enhanced intercellular cross-talk. Aspirin does not counteract prothrombotic effects of exercise. Adenosine is a potentially useful anti-inflammatory agent, but the treatment strategy needs to be optimized to achieve clinically relevant effects.
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| 5. |
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| 6. |
- Sudic, Dzana, et al.
(författare)
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High glucose levels enhance platelet activation : involvement of multiple mechanisms.
- 2006
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Ingår i: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 133:3, s. 315-22
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Tidskriftsartikel (refereegranskat)abstract
- Diabetes mellitus (DM) and hyperglycaemia are associated with platelet activation. The present study was designed to investigate how high glucose levels influence platelet function. Fasting human blood was incubated with different concentrations of D-glucose (5, 15 and 30 mmol/l) and other sugars without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry. High glucose levels enhanced adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced platelet P-selectin expression, and TRAP-induced platelet fibrinogen binding. Similar effects were seen with 30 mmol/l L-glucose, sucrose and galactose. Hyperglycaemia also increased TRAP-induced platelet-leucocyte aggregation. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression, but abolished the enhancement of TRAP-induced platelet fibrinogen binding by hyperglycaemia. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the hyperglycaemic enhancement of platelet P-selectin expression, but did not counteract the enhancement of TRAP-induced platelet fibrinogen binding. Hyperglycaemia did not alter platelet intracellular calcium responses to agonist stimulation. Blockade of cyclo-oxygenase (COX), phosphotidylinositol-3 (PI3) kinase, or nitric oxide synthase, or the addition of insulin did not influence the effect of hyperglycaemia. In conclusion, high glucose levels enhanced platelet reactivity to agonist stimulation through elevated osmolality. This occurred via superoxide anion production, which enhanced platelet P-selectin expression (secretion), and PKC signalling, which enhanced TRAP-induced fibrinogen binding (aggregablity).
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