Hemostatic properties of a venomic protein in rat organ trauma

Previous in vitro work characterized the protease Q8009 isolated from the venom of the Australian brown snake Pseudonaja textilis textilis with Factor Xa-like activity and hemostatic properties. The purpose of the work described here characterizes the in vivo hemostatic properties in a rat model of parenchymatous organ injury. The key parameters of activity included reduction in time-to-hemostasis and total volume of blood loss in spleen, liver and kidney wound models in rats. The surgical protocols involved exposure of the organs via a midline abdominal laparotomy. Using a clean metal template with 6, 6.5, 9 mm holes for spleen, liver and kidney, respectively, a predetermined volume of the organ was gently extruded through the template hole and excised with a razor blade. About 50 to 75 microL of collagen matrix with the different test solutions was applied to the wounds. Blood was collected and at the end of the procedure animals were humanely sacrificed with an anesthetic overdose. Determination of blood was performed using the hematin assay using a standard curve. Blood loss per minute and total blood loss were calculated. Results from the studies demonstrated that the application of Q8009 and collagen matrix to surgical wounds significantly reduced the total amount of blood loss and the time-to-hemostasis. In the spleen wound model, Q8009 at 100, 250 and 1000 microg/ml significantly reduced (p<0.001) the total volume of blood lost relative to thrombin and reduced the time-to-hemostasis by 25-50%, as compared to 7% by thrombin. In the liver wound model, Q8009 at 250 and 1000 microg/ml significantly reduced (p<0.001) the total volume of blood lost relative to thrombin and reduced the time-to-hemostasis from 10.5 min by thrombin to 5.6 min with Q8009. In the kidney wound model, Q8009 at 250 microg/ml significantly reduced (p<0.05) the total volume of blood lost and reduced the time-to-hemostasis by 25% when compared to thrombin. The hemostasis levels were consistent with previous findings in skin wound rat models where Q8009 consistently reduced the total volume of blood lost and shortened time-to-hemostasis. Application of Q8009 plus collagen matrix significantly reduced the volume of total blood loss and time-to-hemostasis in rat surgical organ wound models induced bleeding, as compared to a commercially available hemostat device. The protein Q8009 has greater capacity to reduce blood loss and shorten time-to-hemostasis; highly desirable properties where rapid hemostasis is needed in surgical wounds in parenchymatous organs.