Recombinant annexin A2 plus tPA for combination stroke therapy

描述（由申请人提供）：重组tPA静脉注射仍然是fda批准的唯一且最有效的卒中紧急治疗干预措施。然而，治疗时间窗短、出血转化、溶栓灌注率差以及神经毒性是其应用的主要限制。在本提案中，我们假设重组膜联蛋白A2 （rA2）可以降低再灌注所需的tPA剂量，同时增强溶栓效果，减轻脑出血转化。通过这样做，它将延长治疗时间窗口并改善长期结果。我们的想法的分子基础是膜联蛋白A2通过与tPA和纤溶蛋白前体纤溶酶原组装纤溶蛋白复合物来加速溶凝血纤溶蛋白的产生。我们的试验数据很有希望。我们现在提出三个目标来研究这种新的联合疗法的机制。在Aim 1中，我们将研究局灶性卒中后4小时rA2 +低剂量tPA联合治疗的溶栓效果，MR血管造影来确定再通和再闭塞，MRI CBF来绘制脑血流；以及MRI测量脑血容量（CBV）、微血管体积（MVV）和血管大小指数（VSI），以量化血管对溶栓剂和血管扩张剂的反应。在Aim 2中，我们将研究rA2 +低剂量tPA联合治疗对大鼠局灶性栓塞后4小时神经血管基质蛋白水解和血脑屏障渗漏的影响。脑梗死、水肿和出血转化的发展将被测量，并与MMP-9活性、细胞外基质成分的蛋白水解降解和血脑屏障渗漏的时间谱相关联。在Aim 3中，我们将评估中风后4小时rA2加低剂量tPA联合治疗的长期结果。研究人员将对大鼠进行为期四周的一系列行为测试。最后取脑组织测量H&E病变大小，检测脑血管密度及组织恢复因子VEGF、Ang-1表达。将低剂量tPA加rA2与单独使用rA2、单独使用低剂量和高剂量tPA以及对照盐水进行比较。如果成功，这些实验将为rA2 +低剂量tPA如何改善tPA卒中治疗提供新的见解，并可能最终产生一种新的组合方法来优化基于tPA的溶栓性卒中治疗。我们认为我们的建议应该与PA-07-233“重症监护的多学科转化研究”（R01）直接相关。

DESCRIPTION (provided by applicant): Intravenous administration of recombinant tPA remains the only FDA-approved and the most beneficial proven intervention for emergency treatment of stroke. However, short treatment time window, hemorrhagic transformation, poor thrombolytic perfusion rate, and neurotoxicity comprise the major limitations to the application. In this proposal, we hypothesize that recombinant Annexin A2 protein (rA2) will lower the required dose of tPA for reperfusion, while enhancing thrombolytic efficacy, and attenuating intracerebral hemorrhagic transformation. By doing so, it will prolong therapeutic time windows and improve long-term outcomes. The molecular basis for our idea is that annexin A2 accelerates the generation of clot-dissolving plasmin by assembling a fibrinolytic complex with tPA and the plasmin precursor plasminogen. Our pilot data are promising. We now propose three aims to investigate the mechanisms involved in this new combination therapy. In Aim 1, we will study thrombolytic efficacy of rA2 plus low dose tPA combination treated at 4 hrs after focal embolic stroke in rats with MR angiography to determine recanalization and reocclusion, MRI CBF to map cerebral blood flow; and MRI measurements of cerebral blood volume (CBV), microvascular volume (MVV), and vessel size index (VSI) to quantify vascular responses to thrombolytic agents and vasodilators. In Aim 2, we will investigate effects of rA2 plus low dose tPA combination therapy on neurovascular matrix proteolysis and BBB leakage treated at 4 hrs after focal embolic stroke of rats. The development of brain infarction, edema and hemorrhagic transformation will be measured and correlated with temporal profiles of MMP-9 activity, proteolytic degradation of extracellular matrix components and BBB leakage. In Aim 3, we will assess long term outcomes of rA2 plus low dose tPA combination treated at 4 hrs after stroke. Rats will be followed over 4 weeks with a battery of behavioral tests. At the end, brains are removed for H&E lesion size measurement, cerebral vessel density and tissue recovery factors VEGF, Ang-1 expression will be examined. Low-dose tPA plus rA2 will be compared with rA2 alone, low-dose and high-dose tPA alone, and vehicle saline control. If successful, these proposed experiments should provide new insight of how rA2 plus low dose tPA improve tPA stroke treatment and may ultimately yield a novel combination approach to optimize tPA-based thrombolytic stroke therapy. We believe our proposal should be directly relevant for PA-07-233, "Multidisciplinary Translational Research in Critical Care" (R01).