Microvascular Thrombosis in systemic inflammation

描述（由申请人提供）：弥散性微血管血栓形成，如弥散性血管内凝血（DIC）经常发生在严重的全身性炎症和败血症中。脓毒性DIC患者可能表现为血栓形成前状态，导致突然广泛的微血管血栓形成，阻塞血管。由此产生的微血管阻塞会导致多器官功能衰竭（MOF），并可能导致死亡。死于败血症相关DIC的患者尸检显示器官中广泛存在富含纤维蛋白和血管性血友病因子（VWF）的微血栓。这部分是由于纤维蛋白的大量产生导致纤维蛋白沉积在微血管中，内皮细胞（ECs）分泌的血栓超大型（UL） VWF弦显著增加。这些固定在内皮细胞和沉积的纤维蛋白上的高粘性VWF字符串可以捕获循环血小板，导致微血管闭塞。我们已经确定了vimentin (Vim)，一种也在ECs表面表达的中间丝，作为VWF的配体。Vim和纤维蛋白都与VWF的A2结构域结合。因此，重组A2蛋白（VWF的A2结构域）抑制Vim-和纤维蛋白-VWF的相互作用，阻止炎症内皮细胞上ULVWF串的形成，减少VWF介导的血流依赖性血小板对纤维蛋白的粘附，抑制血小板-纤维蛋白凝块的形成。此外，在内毒素（脂多糖，LPS）损伤1.5小时后，A2蛋白改善了内毒素血症诱导的DIC小鼠模型的血栓形成前状态。在这些小鼠中，A2蛋白增加了存活率，减少了弥散性的富含纤维蛋白和富含vwf的微血栓，阻止了中性粒细胞向肺的浸润，并抑制了促炎细胞因子。最后，在体外lps刺激的ECs中，A2蛋白激活了蛋白磷酸酶2A，降低了PKC和AKT的磷酸化。因此，这项应用探索了新型A2蛋白的治疗潜力，该蛋白可以抵消内皮细胞激活的血栓形成和促炎途径的有害影响。我们提出了三个目标：目标1将研究Vim和纤维蛋白与ULVWF的相互作用如何促进微血管血栓形成；目的2将分析A2蛋白如何抑制lps诱导的ECs信号传导和功能；目的3将确定A2蛋白在猪弥散性微血管血栓形成的MOF模型中的作用。这些研究将揭示Vim在微血管血栓形成中的新作用，以及A2蛋白作为弥散性微血管血栓形成和MOF相关疾病的新治疗剂的潜在用途。

DESCRIPTION (provided by applicant): Disseminated microvascular thromboses such as disseminated intravascular coagulation (DIC) frequently occurs in severe systemic inflammation and sepsis. Septic patients with DIC may manifest a prothrombotic state that lead to a sudden widespread microvascular thrombosis, occluding the vessels. The resultant microvascular occlusion cause multiple organ failure (MOF) and may result in death. Autopsies of patients who succumbed to sepsis-associated DIC demonstrated a widespread fibrin-rich and von Willebrand factor (VWF)-rich microthrombi in organs. This is, in part, because of the massive generation of fibrin that leads to fibrin deposition in the microvasculature and the significant increase in the secretion of the prothrombotic ultra large (UL) VWF strings from endothelial cells (ECs). These hyperadhesive VWF strings anchored on ECs and the deposited fibrin can capture circulating platelets, causing microvascular occlusion. We have identified vimentin (Vim), an intermediate filament that is also expressed on the surface of ECs, as ligand for VWF. Both Vim and fibrin bind to the A2 domain of VWF. Consequently, the recombinant A2 protein (A2 domain of VWF) inhibits Vim- and fibrin-VWF interactions, prevents the ULVWF strings formation on the inflamed ECs, reduces VWF-mediated flow-dependent platelet adhesion to fibrin, and inhibits platelet-fibrin clots formation. Moreover, the A2 protein ameliorated the prothrombotic state in a mouse model of endotoxemia-induced DIC given 1.5 hour after the endotoxin (lipopolysaccharide, LPS) insult. In these mice, the A2 protein increased survival, reduced disseminated fibrin-rich and VWF-rich microthrombi, prevented infiltration of neutrophils into the lung, and dampened proinflammatory cytokines. Lastly, the A2 protein activated protein phosphatase 2A and decreased phosphorylation of PKC and AKT in LPS-stimulated ECs in vitro. Therefore, this application explores the therapeutic potential of the novel A2 protein that counteracts deleterious effects of prothrombotic, and proinflammatory pathways activated at endothelium. We propose three aims: Aim 1 will investigate how interaction of Vim and fibrin with ULVWF contributes to microvascular thrombosis; Aim 2 will dissect how A2 protein dampens LPS-induced signaling and functions in ECs; Aim 3 will determine the effect of A2 protein in porcine models of MOF with disseminated microvascular thromboses. These studies will reveal a novel role for Vim in microvascular thrombosis and the potential utility of the A2 protein as a new therapeutic agent for conditions associated with disseminated microvascular thromboses and MOF.