Thrombin derived from bovine sources commonly is used to arrest bleeding during surgical procedures. However, complications such as postoperative hemorrhage can occur because of the development of cross-reactive anti-bovine antibodies that inhibit human coagulation factor V. It would thus be advantageous to develop techniques to generate human thrombin. This study evaluated thrombin produced from human plasma using a new Thrombin-Processing Device (TPD). Plasma was introduced into the TPD, mixed with an ethanol/ CaCl2 reagent, incubated for 1 h, and the harvested thrombin was assayed for activity and the ability to activate platelets by in vitro assays. TPD-produced thrombin activity was found to be 51.8 +/- 12.4 IU/mL (n = 145). TPD-produced thrombin also stimulated P-selectin (CD62) expression (83 +/- 13% of the platelet population) and Annexin V binding (10.3 +/- 2% of the platelet population) on platelets in a similar fashion to commercial thrombin (P-selectin expression: 88 +/- 3%; Annexin-V binding: 11.4 +/- 3%). Compared with CaCl2 and batroxobin, TPD-produced thrombin had a significantly greater ability to activate platelets. TPD-produced thrombin from human plasma has consistent activity and significantly activates platelets and, thus, may have attractive applications such as the production of autologous thrombin for surgical patients.