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The ability of cellular fibronectin, found in the vessel wall in a fibrillar conformation, to regulate platelet functions and trigger thrombus formation remains largely unknown. In this study, we evaluated how parietal cellular fibronectin can modulate platelet responses under flow conditions. A fibrillar network was formed by mechanically stretching immobilised dimeric cellular fibronectin. Perfusion of anticoagulated whole blood over this surface resulted in efficient platelet adhesion and thrombus growth. The initial steps of platelet adhesion and activation, as evidenced by filopodia extension and an increase in intracellular calcium levels (419 ± 29 nmol/l), were dependent on integrins α5β1 and αIIbβ3. Subsequent thrombus growth was mediated by these integrins together with the GPIb-V-IX complex, GPVI and Toll-like receptor 4. The involvement of Toll-like receptor 4 could be conveyed via its binding to the EDA region of cellular fibronectin. Upon thrombus formation, the platelets became procoagulant and generated fibrin as revealed by video-microscopy. This work provides evidence that fibrillar cellular fibronectin is a strong thrombogenic surface which supports efficient platelet adhesion, activation, aggregation and procoagulant activity through the interplay of a series of receptors including integrins α5β1 and αIIbβ3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.

β-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1-/- and β-arr2-/- platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1-/- but not β-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1-/- and β-arr2-/- mice, suggesting no defect in haemostasis. β-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbβ3. β-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbβ3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in αIIbβ3 signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.