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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 shear rate gradients (SRG) but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create SRG during blood perfusion. VWF-GPIbα interactions were increased at sites of SRG compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRG 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 scFVA1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRG on VWF dependent platelet aggregation and developed the shear gradient-sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of SRG. 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 hemostasis.

Abstract 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.

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.