Abstract: Thrombin is a serine protease important for the mediation and the regulation of the coagulation cascade. The production of thrombin is initiated on the surface of endothelial cells and activated platelets by the formation of the prothrombinase complex. This complex is composed of platelet phospholipids, Ca2+, and coagulation factors Va and Xa, and it proteolytically cleaves prothrombin to thrombin (1). Thrombin is not only critical for the enhancement of coagulation, but it also plays an important role in its control. For example, combined with thrombomodulin present on the endothelial cell surface, thrombin converts protein C to protein Ca. Together with protein S, protein Ca can degrade factors Va and VIIIa, thus limiting the activity of the coagulation cascade. These events are continually ongoing (e.g., at the site of injury) and are responsible for not only reducing blood flow but also promoting wound healing and angiogenesis (2). Thrombin has been used in surgical procedures as a means of reducing wound bleeding for decades. In 1977, Jasani reported that 15 patients treated with topical thrombin after abdominal surgery exhibited significantly fewer hematomas compared with the control group not receiving treatment (3). This finding was later confirmed by Hashemi et al., who showed that heparinized patients receiving treatment with topical thrombin had a reduced rate of hematoma formation (4). Furthermore, thrombin also has been used successfully for the control of bleeding during skin grafting in burn patients (5) and in cardiac surgery (6), among other disciplines. The combination of thrombin with concentrated fibrinogen (cryoprecipitate) and platelets is also a common clinical practice to prevent bleeding and to promote wound healing (7). For example, fibrin sealants (thrombin combined with fibrinogen) are used in a variety of surgical procedures and have been shown to reduce bleeding (8), seroma formation, and drain usage (9). However, thrombin, in combination with platelets is used mainly in orthopedic (10), oral, and maxillofacial surgery (11) for enhancement of bone growth attributable to the increased level of tissue growth factors present in the platelets (12). To date, thrombin used in surgery primarily is produced from bovine plasma sources. A major problem with the use of bovine thrombin is that it can generate the formation of anti-bovine thrombin antibodies (13). These antibodies have been shown to cross-react with the patient’s coagulation Factor V (FV) and cause a range of symptoms, including severe and life-threatening bleeding (13). In 2001, Ortel et al. reported on the risks and prevalence of these adverse reactions and investigated both the formation of antibodies against bovine thrombin and the cross-reactivity against the patient’s FV (14). They found elevated antibody levels against human coagulation factors in 20% of the patients and concluded that bovine thrombin preparations were highly immunogenic and associated with increased risk of adverse outcomes and that re-exposure should be avoided. In addition, the outbreak of bovine spongiform encephalopathy and its eventual linkage to variant Creutzfeldt–Jacob disease in humans (15) has elevated the awareness of substituting bovine products with human, whenever possible. These data suggested that bovine thrombin, although able to reduce bleeding, caused significant adverse events as the result of antibody formation and transmission of disease. To prevent these events, a relatively simple method to produce human thrombin with similar haemostatic properties to commercially available thrombin would be desirable. We developed the Thrombin-Processing Device (TPD™; Thermogenesis Corp, Rancho Cordova, CA), which consists of two parts: a tubular reaction chamber containing a negative surface charge required for initiation of the formation of thrombin and a thrombin reagent consisting of calcium chloride and ethanol. Plasma and thrombin reagent was added to the TPD reaction chamber and the contents were mixed and incubated at room temperature. The TPD was then agitated to break any formed fibrinogen clots and the produced thrombin was ready for harvest. The aim of this study was to assess the activity and platelet-activating ability of the thrombin produced by the TPD and compare it with commercially available thrombin, Ca2+, or batroxobin. Our results show that the TPD-produced thrombin is stable over the course of 6 h and has a similar ability to activate platelets compared with commercial thrombin. The results suggest that the TPD can be used for production of human thrombin from a single donor for autologous use during surgery.