Thrombus formation in vivo is regulated by paracrine signalling with other cells in both the blood and the vessel wall. Thus whilst ex vivo study of isolated human platelet suspensions creates a simplified system in which to assay platelet activation mechanisms, they are unlikely to adequately replicate the responses observed in vivo. Therefore experiments were performed to examine whether exposure of platelet suspensions to simple 3D tissue-engineered constructs of either part or the whole of the blood vessel wall was able to influence the observed calcium signals. Proaggregatory subendothelial matrix constructs were created by coating Poly-lactic acid nanofibres with or without 1µg/cm2 Horm collagen. 3D vessel wall constructs was fabricated by culturing human umbilical vein endothelial cells (HUVECs) on a PLA nanofiber mesh attached atop of a collagen hydrogel in which Human coronary artery smooth muscle cells were cultured. Platelets were isolated from blood obtained by venepuncture of healthy volunteers under informed consent and with local ethical committee approval in accordance with the Declaration of Helsinki. Changes in cytosolic calcium concentration ([Ca2+]cyt) were measured in washed human platelets dually labelled with both Fura-2/AM and DiOC6. Platelet suspensions were exposed to partial or full vessel wall constructs for between 5-15 minutes at 37°C. The vessel construct was then removed to examine platelet adhesion to the surface of the construct by fluorescence microscopy, whilst the calcium signalling capacity of the platelet suspension was examined after stimulated with 0.2U/mL thrombin. Data are presented as mean ± SEM of the number of samples (n) indicated. Statistical significance was tested by Student’s t-test. Exposure of platelets to a proaggregatory subendothelial matrix constructs coated with Horm Collagen significantly potentiated the thrombin-evoked rise in [Ca2+]cyt to 173.7 ±11.7% (n = 4; P < 0.05) of those seen in platelets exposed to an analogous construct made using uncoated nanofibres. This correlated with an increased adhesion of platelets to the collagen-coated nanofibres. Exposure of platelets to the full 3D blood vessel construct elicited a significant reduction in the thrombin-evoked rise in [Ca2+]cyt (36.7 ±11.7% of control; n=6; P < 0.05), when compared to samples not exposed to the construct.The ability of our full blood vessel construct to significantly impair thrombin-evoked rises in [Ca2+]cyt demonstrates the need for ex vivo platelet activation assays to more accurately reflect the in vivo environment, and demonstrates the potential of using tissue-engineered blood vessels constructs to help improve the physiological realism of such studies.