I. Abstract Objectives: Thrombin, a dominant coagulation protease has a vascularization-promoting function. Previous studies showed that the interaction between thrombin and human umbilical vein endothelial cell (HUVEC) results in upregulation in expression of vascular endothelial growth factors (VEGF) receptor. Thrombin signaling in endothelial cells mediated by protease-activated receptors (PARs). Thrombin cleaves the PAR-1, the predominant thrombin receptor in endothelial cells, and results in the tethered ligand; SFLLRN interacts with the extracellular loop of receptor and causes receptor activation. TRAP-6, a synthetic peptide that contains motif SFLLRN, showed agonist activity for thrombin. In this study, we investigated the effect of TRAP-6 on the proliferation of human umbilical vein endothelial cells and adipose-derived stem cells (ASCs). Furthermore, vasculogenesis function of TRAP-6 was determined on human umbilical vein endothelial cells/ adipose-derived stem cells co-culture. Moreover, the ability of TRAP-6 in stimulation of human umbilical vein endothelial cells to express various surface markers was determined. Approach and Results: Here, we used bromodeoxyuridine (BrdU) assay to measure the proliferation of HUVEC and ASC. The results demonstrated that the proliferation of both HUVEC and ASC is enhanced. The HUVEC/ASC co-culture treated with TRAP-6 resulted in denser microvascular network formation in vitro and vascular network parameters such as the number of junctions, the number of tubules and total tubule length are enhanced. Furthermore, immunohistochemically investigation of surface markers in stimulated HUVEC with TRAP-6 using flow cytometry showed slightly increase of E-selectin, CD 73, thrombomodulin and CD 34 expression, decrease in CD 31 expression and no change in CD 146, VE-cadherin, Tie2, and VEGFR2 expression pattern. Conclusion: Our study provides detailed insight into angiogenetic behavior and influence of TRAP-6 on HUVECs. Our data demonstrate TRAP-6 improved either of HUVEC and ASC proliferation with no toxic effect. TRAP-induced HUVEC/ASC co-culture promotes microvascular network formation. However, the HUVEC surface markers do no indiacte significant change after stimulation with TRAP-6. Therefore, we concluded that TRAP-6 has potential in promoting angiogenesis and vascularization in tissue engineering.