Aims: Deep vein thrombosis (DVT) is a prevalent cardiovascular condition. Endothelial-derived extracellular vesicles (EVs) may play a crucial role in platelet-dependent DVT development via platelet activation, but the mechanism is not clear yet. This research aims to understand how platelets and endothelial-derived EVs work in DVT. Methods: The interaction between protein disulfide isomerase (PDI) and glucose-regulated protein 94 (GRP94) was founded by molecular docking. Inferior vena cava stasis–induced mice received PDI and GRP94 inhibitor treatments. Platelet activation, endothelial-derived EVs, and PDI were measured using flow cytometry. The expression of PDI and dimetric GRP94 in platelets co-cultured with hypoxic endothelial cells was confirmed by Western blot or native PAGE. The fluorescence resonance energy transfer assay shows conformational changes in GPIIb/IIIa on platelet surfaces. A tracking experiment was performed using PKH26, which labelled endothelial-derived EVs, and the endocytosis of EVs by platelets was tracked by confocal microscope. Results: In a DVT mouse model, platelets enhance venous thrombus formation in a coagulation-independent manner, instead, platelet activation and the length of the thrombus are related to PDI and GRP94 activity. Next, we found that the expression level of endothelial-derived EVs carrying PDI is significantly increased in plasma. Endothelial-derived EVs carrying PDI are endocytosed by platelets, in which the content of GRP94 dimer is elevated, and consequently increases the expression of surface GPIIb/IIIa. In addition, PDI allosterically interacts with GPIIb/IIIa, which is re-configurated into an activated form. Conclusion: Endothelial-derived EVs carrying PDI induce DVT via interplay with GRP94 and GPIIb/IIIa in platelets. These findings emphasize the significance of platelets in DVT formation, and PDI may be a suitable target in DVT prevention.