Severe Trauma Provokes Pathologic Continuum of Plasmin Activation

项目摘要：严重创伤是造成死亡和残疾的一个重要原因。在恢复期早期，可引起出血、血栓形成和多器官功能障碍综合征；在恢复期后期，它会促进病理性组织修复和体内平衡，从而阻止患者恢复日常生活活动。严重创伤相关死亡和残疾与病理性凝血激活（创伤诱导凝血病（TIC））和炎症（全身性炎症反应综合征（SIRS））的激活程度直接相关，提示减轻TIC和/或SIRS可减少严重创伤引起的并发症。关于严重创伤后TIC和SIRS的分子诱因，存在一个关键的知识缺口。我们的初步数据支持了一个变革性的假设，即纤溶蛋白（纤溶系统的主要蛋白酶，对组织修复和体内平衡至关重要）的不适当的早期激活是引发TIC和SIRS的关键事件，也会导致纤溶蛋白活性的长期丧失，破坏组织修复和体内平衡。前提：在孤立性创伤后，纤溶酶原的激活受到严格调节，并且仅限于伤口部位。然而，在严重创伤后，纤溶酶被全身性激活（高纤溶酶），随后是纤溶酶活性的长期缺陷（低纤溶酶），这两种情况都与不良预后相关。我们的中心假设是：(1)严重创伤后的早期高纤溶是TIC和SIRS的主要促进因素，(2)早期高纤溶通过耗尽纤溶酶原导致低纤溶，以及(3)获得性纤溶酶原缺乏是病理组织稳态和修复的驱动因素。方法与途径：采用小鼠烧伤作为严重创伤的代表性模型，我们将在Aim 1中确定早期高纤溶是否会加速TIC和SIRS，在Aim 2中确定早期高纤溶是否会导致晚期持续的低纤溶。纤溶酶活性将被药理学抑制/增强，并使用新的分子工具进行测量。TIC和SIRS将通过一系列已建立的生物标志物、血小板功能和器官特异性NF-κB定量作为多器官功能障碍综合征的替代测量来评估。纤维蛋白溶解系统将通过量化其单个元素、蛋白酶抑制剂复合物、纤维蛋白降解产物和活性测定来评估。接下来，在Aim 3中，我们将把小鼠烧伤模型与股骨骨折和骨骼肌损伤模型结合起来，评估晚期低纤溶是否会导致骨相关病理；特别是骨折愈合受损，肌肉异位骨化和创伤性骨质疏松症。在分子水平上，我们将确定恢复纤溶酶活性是否能预防这些骨并发症，以及骨病理过程在多大程度上是由纤维蛋白或纤维蛋白介导的炎症引起的。综上所述，如果属实，这些发现将支持以下观点：1)在恢复期早期抑制纤溶酶原激活，以部分预防TIC和SIRS； 2)在恢复期后期复苏纤溶酶原，或通过其他方法促进纤维蛋白溶解，以保持骨骼健康。

Project Summary: Severe trauma is a significant cause of death and disability. Early in convalescence, it causes bleeding, thrombosis and multi-organ dysfunction syndrome; later in convalescence, it instigates pathologic tissue repair and homeostasis, which prevents return to activities of daily living. Severe trauma related death and disability is directly correlated with the degree of activation of pathologic activation of coagulation (trauma- induce coagulopathy (TIC)) and inflammation (systemic inflammatory response syndrome (SIRS)) suggesting that mitigating TIC and/or SIRS would reduce complications caused by severe trauma. There is a key knowledge gap regarding the molecular instigators of TIC and SIRS following severe trauma. Our preliminary data support a transformative hypothesis that implicates inappropriate early activation of plasmin, the principle protease of the fibrinolytic system essential for tissue repair and homeostasis, as a key event that initiates TIC and SIRS, that also results in a prolonged loss of plasmin activity that disrupts tissue repair and homeostasis. Premise: Following an isolated trauma, plasminogen activation is tightly regulated and restricted to the wound site. However, following a severe trauma, plasmin is systemically activated (hyperfibrinolysis) followed by a prolonged deficit of plasmin activity (hypofibrinolysis), both of which are associated with poor outcomes. Our central hypothesis is that (i) early hyperfibrinolysis following severe trauma is a primary accelerant of TIC and SIRS, (ii) early hyperfibrinolysis causes hypofibrinolysis by exhausting plasminogen, and that (iii) the acquired plasminogen deficiency is a driver of pathologic tissue homeostasis and repair. Methods & Approach: Employing a murine burn injury as a representative model of severe trauma, we will determine in Aim 1 whether early hyperfibrinolysis accelerates TIC and SIRS and in Aim 2 whether early hyperfibrinolysis causes late sustained hypofibrinolysis. Plasmin activity will be pharmacologically inhibited/enhanced and measured using novel molecular tools. TIC and SIRS will be assessed with serial analysis of established biomarkers, platelet function, and organ specific NF-κB quantification as a surrogate measure of multiorgan dysfunction syndrome. The fibrinolytic system will be assessed by quantifying its individual elements, protease-inhibitor complexes, fibrin degradation products, and activity assays. Next, in Aim 3 we will combine the murine burn model with a femur fracture and skeletal muscle injury model to assess whether late hypofibrinolysis causes bone-related pathologies; specifically impaired fracture healing, heterotopic ossification in muscle, and trauma-induced osteoporosis. At the molecular level, we will determine if restoring plasmin activity prevents these bone complications and to what extent of the bone pathologic processes are due to fibrin, or fibrin mediated inflammation. Taken together, if true, these findings would provide support for 1) inhibition of plasminogen activation during the early convalescence to prevent, in part, TIC and SIRS and 2) resuscitation of plasminogen, or alternative means of promoting fibrinolysis, during late convalescence, to preserve bone health.