Microvascular Thrombosis in systemic inflammation

描述(申请人提供)：弥散性微血管血栓，如弥散性血管内凝血(DIC)，常见于严重的全身炎症和败血症。患有DIC的脓毒症患者可能表现为血栓前状态，导致突然广泛的微血管血栓形成，阻塞血管。由此产生的微血管闭塞会导致多器官衰竭(MOF)，并可能导致死亡。对死于败血症相关DIC的患者的尸检显示，器官中广泛存在富含纤维蛋白和富含von Willebrand因子(VWF)的微血栓。这在一定程度上是因为大量产生的纤维蛋白导致微血管中的纤维蛋白沉积，以及内皮细胞(ECs)分泌的血栓前超大型(UL)VWF显著增加。这些锚定在内皮细胞和沉积的纤维蛋白上的超粘性VWF线可以捕获循环中的血小板，导致微血管闭塞。我们已经确定Vimentin(Vim)是VWF的配体，它是一种中间细丝，也表达在内皮细胞表面。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，防止炎症内皮细胞上ULVWF串的形成，减少VWF介导的流动依赖的血小板与纤维蛋白的黏附，并抑制血小板-纤维蛋白凝块的形成。此外，A2蛋白改善了内毒素(内毒素)诱导的DIC小鼠模型的血栓前状态，内毒素(内毒素)刺激后1.5小时给予。在这些小鼠中，A2蛋白提高了存活率，减少了弥漫的富含纤维蛋白和VWF的微血栓，防止了中性粒细胞向肺内的渗透，并抑制了促炎细胞因子。A2蛋白可激活脂多糖刺激的内皮细胞蛋白磷酸酶2A，降低PKC和AKT的磷酸化水平。因此，这项应用探索了新型A2蛋白的治疗潜力，该蛋白可以抵消血管内皮细胞激活的血栓前和促炎通路的有害影响。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；目标2将剖析A2蛋白如何抑制内皮细胞内毒素诱导的信号和功能；目标3将确定A2蛋白在猪弥漫性微血管血栓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蛋白作为一种新的治疗播散性微血管血栓和多器官功能衰竭相关疾病的潜在用途。

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.