The Mechanistic Investigation Of Brain-derived Microparticles On The Formation Of Traumatic Brain Injury Associated Coagulopathy

Objective:At present, there is no clear mechanism and effective treatment of traumatic brain injury (TBI) associated coagulopathy in the worldwide. In our experiment, we will establish mouse TBI model, observe the level of brain-derived microparticles (BDMP) and coagulation changes in plasma. Basing on the method of preparing BDMP in vitro, we investigate the procoagulant effect of BDMP, the interactions between BDMP and platelets, and the scavenging effect of Lactadherin on BDMP. We attempt to clarify the material basis of TBI-associated coagulopathy, confirm the mechanism of TBI-associated coagulopathy, and explore a new method to prevent and treat the TBI-associated coagulopathy.Methods:1) Basing on the mouse TBI model, we test the BDMP level and coagulation changes in plasma by flow cytometry and activated factor X clotting time test;2) Preparation of BDMP in vitro by using brain tissue homogenate;3) Identification the morphology and phenotype of BDMP by transmission electron microscopy (TEM), flow cytometry and Western Blot;4) Test the procoagulant effect of BDMP in vitro by activated factor X clotting time and thrombin generation tests;5) After injecting BDMP to normal mice via tail vein, we test the procoagulant effect of BDMP in vivo by activated factor X clotting time test, plasma fibrinogen levels and pathological PTAH staining methods;6) Observation the binding between BDMP and platelets by flow cytometry and scanning electron microscopy;7) Test the activated effect of BDMP on platelets by flow cytometry;8) Test the platelet-mediated migration of BDMP by Transwell chamber system;9) Test the scavenging effect of Lactadherin on BDMP by flow cytometry and activated factor X clotting time assay.Results:1) The level of BDMP in TBI mice plasma shows an initial up-regulation and subsequent reduction trend, and peaks at3h after TBI. The platelet poor plasma (PPP) of mice after TBI3h has significantly shorter clotting time, at the same time it has the highest expression of BDMP. After ultracentrifugation to remove BDMP, the clotting time of MP-free plasma (MPFP) is prolonged;2) BDMP has integral membrane structure and expresses a large number of glial fibrillary acidic protein (GFAP) on the surface under TEM. Flow cytometry and Western Blot show that BDMP expresses a large number of neurons and glial cell-specific markers, without expression of platelets, red blood cells, white blood cells and endothelial cells markers. The PS and TF on the BDMP surface have the procoagulant effects in vitro. Finally, PPP of BDMP injection mice has a prolonged clotting time and significantly decreased fibrinogen level. And fibrin depositions are observed in many tissues;3) BDMP can combine with platelets, and their combination can promote the expression of PS on the platelets surface and activate platelets, leading to increase the expression of platelet activation marker (P-selectin) and calcium influx. In turn, activated platelets can mediate BDMP penetrate activated HUVEC cells;4) Lactadherin can bind BDMP and weaken the procoagulant effect of BDMP.Conclusion:1) We detect BDMP in the plasma of TBI mice, which has procoagulant activity. BDMP may be the material basis of TBI-associated coagulopathy;2) We acquire BDMP in vitro by using brain tissue homogenate. This method can provide enough materials for subsequent experiments in vitro;3) BDMP can activate platelets, and activated platelets can help BDMP penetrate the BBB. These can partly explain the mechanism of TBI-associated coagulopathy;4) BDMP can combine Lactadherin. This scavenging mechanism on BDMP can provide a new idea for the treatment of TBI-associated coagulopathy.