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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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, α-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 α-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.

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. Populärvetenskaplig sammanfattningVåra celler är i konstant behov av syre och...