Deep vein thrombosis is a life-threatening disease that takes millions of people's lives worldwide. Given both technical and ethical issues of using animals in research, it is necessary to develop an appropriatein vitromodel that would recapitulate the conditions of venous thrombus development. We present here a novel microfluidics vein-on-a-chip with moving valve leaflets to mimic the hydrodynamics in a vein, and Human Umbilical Vein Endothelial Cell (HUVEC) monolayer. A pulsatile flow pattern, typical for veins, was used in the experiments. Unstimulated human platelets, reconstituted with the whole blood, accumulated at the luminal side of the leaflet tips proportionally to the leaflet flexibility. Platelet activation by thrombin induced robust platelet accrual at the leaflet tips. Inhibition of glycoprotein (GP) IIb-IIIa did not decrease but, paradoxically, slightly increased platelet accumulation. In contrast, blockade of the interaction between platelet GPIbαand A1 domain of von Willebrand factor completely abolished platelet deposition. Stimulation of the endothelium with histamine, a known secretagogue of Weibel-Palade bodies, promoted platelet accrual at the basal side of the leaflets, where human thrombi are usually observed. Thus, platelet deposition depends on the leaflet flexibility, and accumulation of activated platelets at the valve leaflets is mediated by GPIbα-VWF interaction.