Development Of Hemostatics And Research On The Surface Functional Proteins

Uncontrollable hemorrhage is the leading cause of death in battlefields, vehicle accidents, street violence, wilderness accidents etc. The development of a high-efficiency life-saving hemostatic can effectively reduce the mortality due to hemorrhage. However, large numbers of commercial hemostatic can not meet the requirements of high biocompatibility, quick clotting, ease of use etc. Since2002zeolite hemostatic Quikclot has saved hundreds of lives in battlefields, while there is severe exothermic reaction for Quikclot. The hemostatic efficiency will also decrease when reduce its exothermic reaction by pre-hydration, which greatly limited its application. Therefore, it is an urgent problem to develop a high-efficiency hemostatic with lower side effect. On the other hand, the coagulation mechanism of Quikclot is still unknown although it has been used over ten years. Recently, the development in nanomaterials-biological fluid interaction suggested that the material surface would be covered by a HPC (hard protein corona) after entering into the biological fluid and it was the HPC lead to the main biologic identity of the materials. However, the relationship between the functional proteins in the HPC to the biological impact of the materials is still unclear. The research on the relationships between the functional proteins in the HPC of zeolite surface and the hemostatic efficiency of zeolite not only helps to reveal the underlying procoaglant mechanism of zeolite hemostatic, but also plays critical roles in clarifying the relationship between the HPC and the biological activity of the materials.This dissertation mainly focused on the development of life-saving hemostatic materials, hemostatic mechanism and functional proteins in the HPC over the material surface.In chapter2, we focused on developing high-efficiency hemostatic with low side effect. We studied the effect of ion exchange and group modification on the in vitro coagulation efficiency of Jinyun natural zeolite, and evaluated its hemostatic efficiency in the lethal groin injury model of rabbit, cell toxicity, body toxicity and exothermic reaction. The results show that the ion exchange and group modification can not significantly improve the coagulation efficiency. The natural zeolite can significantly reduce the bleeding time, increase the survival of animals, reduce the exothermic effect and favor the wound healing process. Meanwhile, natural zeolite has good biocompatibility, non-cytotoxicity, and non-systemic toxicity, which make natural zeolite as a promising hemostatic. At the same time, we also developed other natural hemostatic with good clotting efficiency and low exothermic effect, such as montmorillonite and bentonite.In chapter3, we studied the procoagulant activity and thrombin activity of Ca-zeolite/HPC. It was found that there would be formed a HPC with high thrombin activity and high-procoagulant activity on the surface of Ca-zeolite when it was incubated in plasma solution, while limited thrombin activity was observed on other materials or other cations exchange zeolite. Immunoassays and mass spectrometry analysis show that it is the thrombin in the Ca-zeolite/HPC that leads to the high procoagulant activity of Ca-zeolite. The thrombin in the Ca-zeolite/HPC will be inhibited by antithrombin to form a thrombin-antithrombin complex when removing the calcium ions in the zeolite by EDTA. The stability assay of Ca-zeolite/HPC shows that the thrombin activity is stable over a30months storage at room temperature. Finally, we studied the hemostatic efficiency of Ca-zeolite/HPC in a tail bleeding model of hemophilia mouse (Factor VIII deficiency). The results shows that the mice can effectively stop bleeding within4.5min after the application of Ca-zeolite/HPC, but it will still bleed after1h for the Quikclot group. In this chapter we first revealed the underlying procoagulant mechanism of zeolite and clarified the relationship between the procoagulant activity of zeolite and HPC.Traditional Chinese medicine (TCM) is a great treasure of our country. Many TCM also have procoagulant effect, however the coagulation mechanism and influencing factors are still unknown. Therefore, we studied the influence factors on the hemostatic efficiency and hemostatic mechanism of ophicalcite in chapter4. The results show that the producing area and processing methods play critical roles in the hemostatic efficiency of ophicalcite. The calcium acetate formed in the processing process contributed to the acceleration of the blood coagulation. In certain range, the thrombin activity and thrombin adsorption on the surface of calcite increase as the increase in the formation of calcium acetate. The study reveals the underlying procoagulant mechanism of ophicalcite and favors the application of TCM.In chapter5, we studied the surface group mediated thrombin generation and adsorption in the HPC of SBA-15. It was found that the negatively charged groups (-OH,-SH,-COOH) could promote the blood clotting of SBA-15, while positively charged group (-NH2) inhibit the blood coagulation. More important, the surface groups play critical roles in the HPC formation and thrombin activity in the HPC:-OH and-SH group modified SBA-15show moderate adsorption to BSA and leads to more thrombin absorbed in the HPC, thus the HPC show high thrombin activity.-COOH modified SBA-15show strong adsorption to BSA and leads to the weak interaction between thrombin, thus most of the thrombin in the HPC was inhibited by anti-thrombin. The research reveals the effects of the surface groups on the adsorption of the functional protein in the HPC.In Chapter6, we studied the effect of crystal phase of titanium dioxide on the HPC formation and the activity of the adsorbed thrombin. The results show that anatase has weak affinity to the plasma proteins and leads to less protein in the HPC, while rutile has strong affinity to the plasma proteins and forms a HPC with much more proteins. The results of thrombin activity on the titanium dioxide surface reveal that there is a sharp decrease in thrombin activity after adsorption to rutile surface. On the other side, the thrombin activity does not show significant change after adsorption to anatase surface. The research clarified the effect of crystal phase on the HPC formation and the thrombin activity on the surface of titanium dioxide, and played a critical role for the biological applications titanium dioxide.Finally, we summarized the dissertation, and made an outlook for the development trend of hemostatic materials and material-HPC interactions in Chapter...