Platelet Activation in Vascular Disease: from Animal Studies to Clinical Consequences

The zebrafish is an in vivo model system originally used to study development of vertebrates. Over the last years it has gained a more important role in studies focusing on diseases such as cancer and thrombosis. Platelets play an important role in thrombosis and therefore drugs inhibiting platelet enzymes and receptors are developed to prevent thrombosis. The most effective drugs inhibit the COX enzymes (aspirin) or the platelet activating receptor P2Y-12 (clopidogrel among others). Lack of clarity exists about the P2Y12 receptor. In T2DM patients, there is speculation that there is less inhibition by 16 antagonists. Also the current insight in P2Y12 signaling is limited to suppression of production of cAMP (a platelet inhibitor) and activation of protein kinase B/Rap1b (which stimulate aggregation). Aims of the thesis: (1) To evaluate the position of the zebrafish as a model system in research on platelet function and thrombus; (2) Discuss the main problems the currently most used anti-platelet drugs (aspirin and clopidogrel) possess; (3) To study the inhibition of the P2Y-12 receptor in diabetes patients and to demonstrate which secondary signalling molecules are activated by P2Y-1

Construct validity of the LapSim: can the LapSim virtual reality simulator distinguish between novices and experts?

BACKGROUND: Virtual reality simulators may be invaluable in training and assessing future endoscopic surgeons. The purpose of this study was to investigate if the results of a training session reflect the actual skill of the trainee who is being assessed and thereby establish construct validity for the LapSim virtual reality simulator (Surgical Science Ltd., Gothenburg, Sweden). METHODS: Forty-eight subjects were assigned to one of three groups: 16 novices (0 endoscopic procedures), 16 surgical residents in training (>10 but 100 endoscopic procedures). Performance was measured by a relative scoring system that combines single parameters measured by the computer. RESULTS: The higher the level of endoscopic experience of a participant, the higher the score. Experienced surgeons and surgical residents in training showed statistically significant higher scores than novices for both overall score and efficiency, speed, and precision parameters. CONCLUSIONS: Our results show that performance of the various tasks on the simulator corresponds to the respective level of endoscopic experience in our research population. This study demonstrates construct validity for the LapSim virtual reality simulator. It thus measures relevant skills and can be integrated in an endoscopic training and assessment progra

Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation

BACKGROUND: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. METHODS AND RESULTS: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos. CONCLUSION: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.

Mlck1a is expressed in zebrafish thrombocytes and an essential component for thrombus formation

Summary Background: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate Myosin Light Chain Kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. Methods and Results: Here we report that Mlck1a is an essential component for thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we, compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5 fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77 fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a deficient embryos. Conclusion: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.

Mlck1a Is Expressed in Zebrafish Thrombocytes and Is an Essential Component of Thrombus Formation

Background: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate Myosin Light Chain Kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. Methods and Results: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos. Conclusion: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.Stem Cell and Regenerative Biolog