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Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of SRGs but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create shear rate gradients during blood perfusion. VWF-GPIbα interactions were increased at sites of shear rate gradients compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRGs but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFV-A1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRGs on VWF dependent platelet aggregation and developed the shear-gradient sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of shear rate gradients. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal haemostasis.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules. The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

Objective: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets’ inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s−1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. Conclusions: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Cardiovascular diseases (CVDs) are the number one cause of death worldwide. The majority of CVD-related deaths are due to heart attacks or stroke, which are often instigated by thrombosis, i.e. the formation of blood clots inside of blood vessels. Therefore, both the academic world and industry seek to better understand, prevent and treat CVDs and thrombosis. However, the number of CVD-related deaths are still rising. Furthermore, the number of therapeutics in the drug development pipeline has stagnated and is disproportionately lower for CVDs in comparison to diseases with a lower burden on society, e.g. cancer or infectious diseases. The development of novel therapeutics is a long and expensive process, currently taking twelve to fifteen years and over one billion euros per new drug. At present, 80% of all drug candidates still fail in the clinical trials, often due to a lack of efficacy or unexpected side-effects. Regardless of the reason for termination, the high rates of late stage failure indicate a poor predictive power of the pre-clinical in vitro and animal models. Therefore, pre-clinical models that can reliably mimic human physiology and disease pathophysiology to predict the effects of therapeutics in humans are needed. Organ-on-a-chip devices could be the answer to the need for technology that can mimic human response. Organ-on-a-chip technology relies on microfluidic technology to provide a realistic environment for human cells to be cultured. Furthermore, patient-specific cells can be incorporated which allows for a personalized approach. The use of patient-specific cells could improve the prediction of efficacy and toxicity during pre-clinical studies. Furthermore, it could lead to the implementation of a personalized medicine approach where a specific drug combination and dose is tailored for each patient. These microfluidic organ-on-chip devices have micrometer-size channels in which human cells can be cultured. Furthermore, similar biochemical cues can be applied as in standard in vitro tests but with the added benefit of control over physical properties like fluid flow and disease-specific geometries. The aim of this PhD project was to develop new thrombosis-on-a-chip devices that can more realistically mimic human physiology and disease pathophysiology. To this end, various possible improvements were investigated with a focus on: realistic disease-specific geometries, the incorporation of hiPSC-ECs, a robust data analysis methodology and an approach to locally activate endothelium. These improvements to thrombosis-on-a-chip devices will bring them closer to being used routinely as pre-clinical models in the drug development pipeline. Furthermore, by incorporating person-specific cells, the models could potentially be utilized for future studies in personalized medicine.

We demonstrate an in vitro microfluidic cell culture platform that consists of periodic 3D hydrogel compartments with controllable shapes. The microchip is composed of approximately 500 discontinuous collagen gel compartments locally patterned in between PDMS pillars, separated by microfluidic channels. The typical volume of each compartment is 7.5 nanoliters. The compartmentalized design of the microchip and continuous fluid delivery enable long-term culturing of Caco-2 human intestine cells. We found that the cells started to spontaneously grow into 3D folds on day 3 of the culture. On day 8, Caco-2 cells were co-cultured for 36 hours under microfluidic perfusion with intestinal bacteria (E. coli) which did not overgrow in the system, and adhered to the Caco-2 cells without affecting cell viability. Continuous perfusion enabled the preliminary evaluation of drug effects by treating the co-culture of Caco-2 and E. coli with 34 µg ml−1 chloramphenicol during 36 hours, resulting in the death of the bacteria. Caco-2 cells were also cultured in different compartment geometries with large and small hydrogel interfaces, leading to differences in proliferation and cell spreading profile of Caco-2 cells. The presented approach of compartmentalized cell culture with facile microfluidic control can substantially increase the throughput of in vitro drug screening in the future.

Arterial thrombosis is the main instigating factor of heart attacks and strokes, which result in over 14 million deaths worldwide every year. The mechanism of thrombosis involves factors from the blood and the vessel wall, and it also relies strongly on 3D vessel geometry and local blood flow patterns. Microfluidic chip-based vascular models allow controlled in vitro studies of the interaction between vessel wall and blood in thrombosis, but until now, they could not fully recapitulate the 3D geometry and blood flow patterns of real-life healthy or diseased arteries. Here we present a method for fabricating microfluidic chips containing miniaturized vascular structures that closely mimic architectures found in both healthy and stenotic blood vessels. By applying stereolithography (SLA) 3D printing of computed tomography angiography (CTA) data, 3D vessel constructs were produced with diameters of 400 μm, and resolution as low as 25 μm. The 3D-printed templates in turn were used as moulds for polydimethylsiloxane (PDMS)-based soft lithography to create microfluidic chips containing miniaturized replicates of in vivo vessel geometries. By applying computational fluid dynamics (CFD) modeling a correlation in terms of flow fields and local wall shear rate was found between the original and miniaturized artery. The walls of the microfluidic chips were coated with human umbilical vein endothelial cells (HUVECs) which formed a confluent monolayer as confirmed by confocal fluorescence microscopy. The endothelialised microfluidic devices, with healthy and stenotic geometries, were perfused with human whole blood with fluorescently labeled platelets at physiologically relevant shear rates. After 15 minutes of perfusion the healthy geometries showed no sign of thrombosis, while the stenotic geometries did induce thrombosis at and downstream of the stenotic area. Overall, the novel methodology reported here, overcomes important design limitations found in typical 2D wafer-based soft lithography microfabrication techniques and shows great potential for controlled studies of the role of 3D vessel geometries and blood flow patterns in arterial thrombosis.