COVID-19 has been associated with an active immunothrombotic process. The involvement of endothelial cells (ECs) in the feedback loop of the inflammatory and thrombotic process characteristic of COVID-19, as well as its differences with other infectious inflammatory conditions, remains an area requiring further elucidation. This study aimed to assess the immunothrombotic phenotype induced by the SARS-CoV-2 Spike (S) protein and its receptor-binding domain (RBD) in endothelial-derived cell lines. HUVEC and EA.hy926 cell lines were exposed to S protein and to its RBD. Inflammatory, thrombotic, and fibrinolytic mediators were quantified. Molecular docking assays were conducted to identify potential EC receptors for S protein. EC activation was dependent on both protein concentration and stimulation time. An increased release of immunothrombotic biomarkers were observed in endothelial-derived cells exposed to the S protein and to its RBD. The RBD induced a stronger endothelial response. Molecular docking demonstrated high affinity and a possible interaction between the S protein and endothelial receptors: CD-141, CD-147, IL-6R, TLR 2, 4, and 7. These findings confirm that the S protein and its RBD can induce an immunothrombotic phenotype in EC-derived cell lines, potentially exacerbating the disease pathology. We propose possible endothelial receptors mediating this response.