Investigation Of The Mechanism Of Brain-derived Mitochondrial Microparticles In Traumatic Brain Injury-associated Coagulopathy

Objective: Secondary cerebral injuries caused by traumatic brain injury(TBI)-associated severe coagulopathy are the major causes of death or disability. However, the mechanistic link between immune inflammation/coagulopathy and cerebral injures secondary to TBI remains poorly understood. We have recently reported for the first time that traumatically injured brain released brain-derived cellular microparticles(BDMP) that synergized with platelets to disrupt the blood brain barrier and to release themselves into the systemic circulation. These BDMP are highly procoagulant for their high expressions of tissue factor and anionic phospholipids, resulting in systemic consumptive coagulopathy and secondary brain injuries in mouse models of TBI. Based on the previous study, we futher clarity the subset of BDMP and their effects on TBI-associated coagulopathy.Methods:(1) We will detect anti-brain antibodies after traumatic brain injury mice model.(2) Detecting the procoagulant effect of cardiolipin(CL) in vitro and in vivo and identify the micelle formation of CL under scaning electron microscope(SEM).(3) Detecting mt MP in peripheral blood after TBI by flow cytometry, RT-PCR and transmission electron microscopy(TEM). Testing the relationship of CL and mt MP in TBI.(4) Isolating mt MP by flow cytometry, immune-beads and multi-step centrifuge and identity its phenotype and morphology by flow cytometry, TEM.(5) Testing the procoagulant effect of mt MP in vitro and in vivo by using acitivating factor Xa clotting time assay, D-dimer and histological staining.(6)Investigating the mt MP releasing from cultured neuron and glia cell lines by flow cytometry and RT-PCR.(7) Detecting whether or not mt MP or CL can activating platelet and its mechanism.(8)Detecting the effects of mt MP or CL on endothelium cells barrier integrity and activation.(9)Detecting the inhibition effect of Lactadhein on mt MP induced procogulant in vitro.Results:(1) We report the detection of CL-exposed brain-derived mitochondrial microparticles(mt MPs) at 17,547±2,677/μl in the peripheral blood of mice subjected to fluid percussion injury to the brain. These mt MPs accounted for 55.2±12.6% of all plasma annexin V-binding microparticles found in the acute phase of injury. Theywere also released from cultured neuronal and glial cells undergoing apoptosis. The mt MPs synergized with platelets to facilitate vascular leakage by disrupting the endothelial barrier.(2) The disrupted endothelial barrier also allowed the release of mt MPs into the systemic circulation to promote coagulation in both traumatically injured and mt MP- or CL-injected mice, leading to enhanced fibrinolysis, vascular fibrin deposition and thrombosis.(3) This mt MP-induced coagulation was mediated by CL transported from the inner to the outer mitochondrial membrane and was blocked by the scavenging molecule lactadherin. The mt MP-bound CL was ~1,600 times as active as purified CL in promoting coagulation.Conclusions:(1) Mitochondria were released from traumatically injured brain into systemic circulation and exposed cardiolipin on their surface(2) Cardiolipin-exposed mitochondria are highly procoagulant and induced traumatic brain injury-associated coagulopathy. This study uncovered a novel procoagulant activity of CL and CL-exposed mitochondria that may contribute to traumatic brain injury-associated coagulopathy and identified potential means of blocking this activity.