liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2018 (English)Doctoral 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.  

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. , p. 54
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1639
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:liu:diva-150960DOI: 10.3384/diss.diva-150960ISBN: 9789176852231 (print)OAI: oai:DiVA.org:liu-150960DiVA, id: diva2:1245952
Public defence
2018-10-05, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2019-09-30Bibliographically approved
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, 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: 2018-09-06
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, 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: 2019-11-27Bibliographically 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: 2018-09-06

Open Access in DiVA

Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation(1602 kB)165 downloads
File information
File name FULLTEXT01.pdfFile size 1602 kBChecksum SHA-512
ae55e433e441f6b633a1b9b38c96fffa9b4fd212ac116f653b5e15a7efe623a3c2c1ccf852749d597a176572651f331963fe2022d5bff0f2856862f4fb49505a
Type fulltextMimetype application/pdf
omslag(5877 kB)10 downloads
File information
File name COVER01.pdfFile size 5877 kBChecksum SHA-512
0059fceb3d73f1624e26e7253093382f38508e4e84023cee223848fdcde8662bc952791af9d856d23175c78ca21c579339f33fcee30a0a2469bb7125dafefb61
Type coverMimetype application/pdf
Order online >>

Other links

Publisher's full text

Authority records BETA

Tunströmer, Kjersti

Search in DiVA

By author/editor
Tunströmer, Kjersti
By organisation
Division of Microbiology and Molecular MedicineFaculty of Medicine and Health Sciences
Biomedical Laboratory Science/Technology

Search outside of DiVA

GoogleGoogle Scholar
Total: 165 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
isbn
urn-nbn

Altmetric score

doi
isbn
urn-nbn
Total: 367 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf