Preparation and characterization of human thrombin for use in a fibrin glue

Cryoprecipitate is frequently combined with thrombin to produce a fibrin sealant to enhance haemostasis during surgical procedures. We evaluated the thrombin produced from plasma using the Thrombin Processing Device (TPD)trade mark (Thermogenesis, Rancho Cordova, CA, USA). Plasma (250 mL) was processed in the CryoSeal FS System using the CP-3 disposable to produce cryoprecipitate by automated freezing and thawing. Simultaneously, thrombin was generated using the attached TPD. The cryoprecipitate and thrombin were harvested after approximately 50 min and then frozen and stored at -80 degrees C until analysis of total protein, fibrinogen, factor VIII (FVIII) activity, von Willebrand factor (vWF) and thrombin activity. Sodium dodecyl sulphate (SDS) gel electrophoresis was used to compare thrombin. After combining the thrombin with cryoprecipitate, the rate of clot initiation and strength was measured using a Thromboelastograph (TEG) (Haemoscope Corp, Skokie, IL, USA). Cryoprecipitate was produced, with a fibrinogen concentration of 22 +/- 7.7 g L(-1) (20 +/- 2% recovery), FVIII activity of 14.2 +/- 4.0 IU mL(-1) and vWF of 19.9 +/- 5.2 IU mL(-1). The separate thrombin product had a concentration of 64.3 +/- 16.7 IU mL(-1) of thrombin and a total protein of 0.39 +/- 0.1 g, with SDS gel electrophoresis showing a major band at 37 kD, as did the commercial human thrombin. The TEG curves of cryoprecipitate and TPD-produced or commercial thrombin were compared. The R values (time to clot initiation) were somewhat slower with the TPD-produced thrombin, but the maximum strength (MA) of the clots was similar. In conclusion, human thrombin can be produced during automated cryoprecipitate production. This thrombin is in sufficient concentration to initiate clotting and cross-linking of fibrin from cryoprecipitate to produce an entirely autologous fibrin glue.