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  • 1.
    Tunströmer, Kjersti
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
    Faxälv, Lars
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Larsson, Pia
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences. Monash Univ, Australia.
    Lindahl, Tomas
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Boknäs, Niklas
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Haematology.
    Thrombus remodelling by reversible and irreversible P2Y(12) inhibitors2023In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 34, no 1, article id 2157805Article in journal (Refereed)
    Abstract [en]

    Pharmacological inhibition of the platelet ADP-receptor P2Y(12) is a cornerstone in the prevention of atherothrombotic events in adult patients with acute coronary syndrome (ACS). Thienopyridines such as clopidogrel and prasugrel exert their antithrombotic effect by means of active metabolites that irreversibly inhibit P2Y(12). Due to the short half-life of these metabolites, a subpopulation of ADP-responsive platelets will form in between dosing. With increased platelet turnover rate or poor patient compliance, the fraction of ADP-responsive platelets will increase, potentially increasing the risk for new thrombotic events. In contrast, the reversible P2Y(12) inhibition produced by direct-acting ADP blockers such as ticagrelor and cangrelor inhibit the entire platelet population. In this study, we evaluated the impact of these pharmacological differences on thrombus formation in an ex vivo flow chamber model. A customized image analysis pipeline was used for automatized, large-scale identification and tracking of single platelets incorporated into the thrombus, enabling quantitative analysis of the relative contribution of inhibited and uninhibited platelets to thrombus growth and consolidation. Comparative experiments were conducted using the irreversible and reversible P2Y(12) inhibitors prasugrel active metabolite (PAM) and ticagrelor, respectively. Our results show that PAM inhibited thrombus platelet recruitment more gradually than ticagrelor, with a slower onset of inhibition. Further, we show that the presence of a small fraction (<10%) of uninhibited platelets did not abrogate the antithrombotic effect of PAM to any significant extent. Finally, we demonstrate a gradual enrichment of inhibited platelets in the thrombus shell due to selective recruitment of inhibited platelets to the thrombus periphery.

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  • 2.
    Lund, Mikael
    et al.
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
    Macwan, Ankit
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
    Tunströmer, Kjersti
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
    Lindahl, Tomas
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Boknäs, Niklas
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Haematology.
    Effects of Heparin and Bivalirudin on Thrombin-Induced Platelet Activation: Differential Modulation of PAR Signaling Drives Divergent Prothrombotic Responses2021In: Frontiers in Cardiovascular Medicine, E-ISSN 2297-055X, Vol. 8, article id 717835Article in journal (Refereed)
    Abstract [en]

    Heparin and bivalirudin are widely used as anticoagulants in the setting of acute thrombosis. In this study, we investigated how these drugs affect the ability of thrombin to generate a prothrombotic platelet response via activation of the protease-activated receptors (PARs) 1 and 4. We examined the effects of heparin/antithrombin and bivalirudin on PAR1- and PAR4-mediated intracellular calcium mobilization, aggregation, alpha-granule release, and procoagulant membrane exposure in platelets exposed to thrombin concentrations likely to be encountered in the thrombus microenvironment during thrombosis. At physiological antithrombin levels, heparin treatment resulted in complete and sustained inhibition of thrombin-induced PAR4-mediated platelet activation, but transient PAR1 signaling was sufficient to elicit significant alpha-granule release and platelet aggregation. In contrast, bivalirudin treatment resulted in rapid and profound inhibition of signaling from both PAR receptors, followed by a delayed phase of PAR4-mediated platelet activation, resulting in a robust prothrombotic response. Combination treatment with bivalirudin and subtherapeutic concentrations of heparin completely inhibited the residual platelet activation observed with single drug treatment at all time-points. Our results show that heparin and bivalirudin have different and complementary inhibitory effects on the activation of PAR1 and PAR4 by thrombin.

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  • 3. Order onlineBuy this publication >>
    Tunströmer, Kjersti
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Blood platelets are a part of the complex system called haemostasis aimed at ensuring our blood’s continuous transport of oxygen and nutrients throughout the body. The transport is ensured by limiting blood loss due to vessel injury and in this process, the platelets form a plug in the damaged area, reinforced by the formation of fibrin. Similar mechanisms may cause thrombus formation, often triggered by atherosclerotic plaque rupture, causing vessel occlusion, embolism or ischemia, which may cause irreversible damage to the heart or the brain.

    Platelet research is crucial for improved prevention and treatment of thrombotic disorders. For such research, flow chambers are an interesting tool for studies of platelet adhesion, aggregation and thrombus formation under similar flow conditions as in the blood vessels, which is important, as the flow affects the mechanisms involved in both haemostasis and thrombosis. Flow chambers can be designed for specific purposes, such as for the study of haemostasis at specific flow conditions or to evaluate drugs or biomaterials. In this thesis, our aim has been to improve the usefulness of in-vitro flow chambers and develop a more robust and informative image analysis of such experiments.

    Initially, we introduced an internal control within each flow chamber experiment, thereby reducing the experimental variance caused by unknown factors. Furthermore, control and sample were thus exposed to identical experimental settings. By using platelet count as quantification of thrombus formation we introduce a method of analysis with increased or similar sensitivity to today’s standards. The platelet count method facilitated comparison of results obtained in different types of flow chambers by an absolute scale of measurement, independent of user settings. The platelet count method was further developed so that additional parameters could be analysed, providing more information about each individual platelet and the overall thrombus. The parameters analysed included platelet stability, height, movement and contraction. The method was used to evaluate how the pharmacokinetics of a reversible (ticagrelor) and irreversible (prasugrel) platelet ADP-receptor inhibitor affected the overall thrombus formation. Especially, how a non-inhibited platelet fraction, formed between drug administrations of irreversible inhibitors, affected thrombus formation. In addition, we sought to understand the regulation of the thrombin receptor, PAR1, expression in cancer cells. We found the microRNA miR20b to be antioncogenic through its downregulation of PAR1 expression.

    This thesis contains numerous flow chamber experiments. However, for further use and full potential of the method increased standardisation is important. Our work regarding the quantification and analysis of flow chamber experiments will contribute to a more robust analysis and maybe even more important, provide new and detailed information on thrombus formation.  

    List of papers
    1. Counting the platelets: a robust and sensitive quantification method for thrombus formation
    Open this publication in new window or tab >>Counting the platelets: a robust and sensitive quantification method for thrombus formation
    2016 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, E-ISSN 2567-689X, Vol. 115, no 6, p. 1178-1190Article in journal (Refereed) Published
    Abstract [en]

    Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, y- and z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.

    Place, publisher, year, edition, pages
    SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN, 2016
    Keywords
    Platelet aggregation; microfluidics; thrombosis; fluorescence microscopy; computer-assisted image processing
    National Category
    Clinical Laboratory Medicine
    Identifiers
    urn:nbn:se:liu:diva-130073 (URN)10.1160/TH15-10-0799 (DOI)000377237400011 ()26842994 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council [K2015-79X-22644-01-3]; Linkoping University

    Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2023-08-28
    2. Quantification of Platelet Contractile Movements during Thrombus Formation
    Open this publication in new window or tab >>Quantification of Platelet Contractile Movements during Thrombus Formation
    2018 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, E-ISSN 2567-689X, Vol. 118, no 09, p. 1600-1611Article in journal (Refereed) Published
    Abstract [en]

    Imaging methods based on time-lapse microscopy are important tools for studying the dynamic events that shape thrombus formation upon vascular injury. However, there is a lack of methods to translate the vast amount of visual data generated in such experiments into quantitative variables describing platelet movements that can be subjected to systematic analysis. In this study, we developed experimental and computational protocols allowing for a detailed mathematical analysis of platelet movements within a developing thrombus. We used a flow chamber-based model of thrombosis wherein a collagen strip was used to initiate platelet adhesion and activation. Combining the use of a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways.

    Place, publisher, year, edition, pages
    New York: Georg Thieme Verlag KG, 2018
    Keywords
    flow chambers, thrombosis, platelet aggregation, platelet contraction, fluorescence microscopy
    National Category
    Medical Image Processing
    Identifiers
    urn:nbn:se:liu:diva-150961 (URN)10.1055/s-0038-1668151 (DOI)000444575200013 ()30112750 (PubMedID)
    Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2023-08-28Bibliographically approved
    3. miR-20b regulates expression of proteinase-activated receptor-1 (PAR-1) thrombin receptor in melanoma cells
    Open this publication in new window or tab >>miR-20b regulates expression of proteinase-activated receptor-1 (PAR-1) thrombin receptor in melanoma cells
    Show others...
    2014 (English)In: Pigment Cell & Melanoma Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 27, no 3, p. 431-441Article in journal (Refereed) Published
    Abstract [en]

    The proteinase-activated receptor 1 (PAR-1) plays a central role in melanoma progression and its expression level is believed to correlate with the degree of cancer invasiveness. Here, we show that PAR-1 is post-transcriptionally regulated by miR-20b microRNA in human melanoma cells. PAR-1 was found to be expressed in metastatic melanoma cells but was barely detectable in primary melanoma. By transducing primary melanoma cells with a lentivirus containing a 3-UTR construct of PAR-1 mRNA, we could show that endogenous melanoma microRNAs interacted with PAR-1 3-UTR and silenced a fused luciferase reporter. Transfection of an inhibitor against miR-20b into primary melanoma cells reversed this process. Finally, transfection of miR-20b mimic into metastatic melanoma cells caused downregulation of the luciferase reporter. We conclude that miR-20b regulates expression of melanoma PAR-1 receptor, which may explain the differential expression of PAR-1 observed in human melanoma.

    Place, publisher, year, edition, pages
    Wiley, 2014
    Keywords
    melanoma; metastasis; proteinase-activated receptor-1; gene expression regulation; microRNAs
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-106844 (URN)10.1111/pcmr.12217 (DOI)000334170900014 ()
    Available from: 2014-05-28 Created: 2014-05-23 Last updated: 2022-04-19
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    Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation
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  • 4.
    Tunströmer, Kjersti
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Faxälv, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Boknäs, Niklas
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Haematology.
    Lindahl, Tomas L.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Quantification of Platelet Contractile Movements during Thrombus Formation2018In: Thrombosis and Haemostasis, ISSN 0340-6245, E-ISSN 2567-689X, Vol. 118, no 09, p. 1600-1611Article in journal (Refereed)
    Abstract [en]

    Imaging methods based on time-lapse microscopy are important tools for studying the dynamic events that shape thrombus formation upon vascular injury. However, there is a lack of methods to translate the vast amount of visual data generated in such experiments into quantitative variables describing platelet movements that can be subjected to systematic analysis. In this study, we developed experimental and computational protocols allowing for a detailed mathematical analysis of platelet movements within a developing thrombus. We used a flow chamber-based model of thrombosis wherein a collagen strip was used to initiate platelet adhesion and activation. Combining the use of a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways.

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    Supplematary Material: Image Processing and Analysis
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    Supplementary Video S1
    Download (mp4)
    Supplementary Video S2
  • 5.
    Claesson, Kjersti
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Faxälv, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Counting the platelets: a robust and sensitive quantification method for thrombus formation2016In: Thrombosis and Haemostasis, ISSN 0340-6245, E-ISSN 2567-689X, Vol. 115, no 6, p. 1178-1190Article in journal (Refereed)
    Abstract [en]

    Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, y- and z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.

    Download full text (pdf)
    fulltext
    Download (mp4)
    film
  • 6.
    Saleiban, Amina
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Faxälv, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Claesson, Kjersti
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Jönsson, Jan-Ingvar
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Osman, Abdimajid
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    miR-20b regulates expression of proteinase-activated receptor-1 (PAR-1) thrombin receptor in melanoma cells2014In: Pigment Cell & Melanoma Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 27, no 3, p. 431-441Article in journal (Refereed)
    Abstract [en]

    The proteinase-activated receptor 1 (PAR-1) plays a central role in melanoma progression and its expression level is believed to correlate with the degree of cancer invasiveness. Here, we show that PAR-1 is post-transcriptionally regulated by miR-20b microRNA in human melanoma cells. PAR-1 was found to be expressed in metastatic melanoma cells but was barely detectable in primary melanoma. By transducing primary melanoma cells with a lentivirus containing a 3-UTR construct of PAR-1 mRNA, we could show that endogenous melanoma microRNAs interacted with PAR-1 3-UTR and silenced a fused luciferase reporter. Transfection of an inhibitor against miR-20b into primary melanoma cells reversed this process. Finally, transfection of miR-20b mimic into metastatic melanoma cells caused downregulation of the luciferase reporter. We conclude that miR-20b regulates expression of melanoma PAR-1 receptor, which may explain the differential expression of PAR-1 observed in human melanoma.

1 - 6 of 6
CiteExportLink to result list
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  • oxford
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