OC 49.3 - A microfluidic model to study the initiation of venous thrombosis

Background: Venous thromboembolism is the third highest cause of cardiovascular mortality. Low/aberrant flow in the venous valves is a major contributing risk factor in the development of thrombosis. Although both platelets and neutrophils are important in the initiation of venous thrombi in valve pockets, underlying mechanisms remain to be elucidated.

Aims: To develop an in vitro model to investigate the initiating events leading to thrombus formation in venous valves.

Methods: Primary HUVECs were cultured in the presence or absence of shear, either in straight channels, or in microchannels with a shape approximating pathogenic venous valves. Expression levels of haemostatic markers were quantified using qPCR, Western blot or immunostaining. Live imaging flow assays in endothelialised valve channels were used to quantify the recruitment of platelets and neutrophils.

Results: ECs cultured under static conditions (i.e. in venous pockets) showed significantly higher expression of VWF, EPCR and TFPI compared with ECs cultured under venous shear, while TM was downregulated at both mRNA and protein level. Within valve channels, ECs cultured in the linear section showed alignment in the direction of flow, whereas those in the venous pocket retained a cobblestone morphology, highlighting the different shear stresses present in each region. Flow assays using plasma-free blood revealed the ability of neutrophils to bind to platelets/ECs specifically in the valve pocket, but not in the linear section. Furthermore, neutrophils captured in the pocket region formed NETs within 60 mins.

Conclusion(s): Our data demonstrate the influence of flow on EC phenotype and subsequent capture and activation of platelets and neutrophils. Further experiments are under way to elucidate key receptors and signalling pathways involved in this process. Our fluidic venous valve model will be invaluable to investigate key initiating events leading to neutrophil capture on platelets and/or ECs and may highlight novel targets for therapeutic intervention.