Recombinant annexin A2 plus tPA for combination stroke therapy

描述(申请人提供)：静脉注射重组tPA仍然是FDA批准的唯一和最有益的已证实的中风紧急治疗干预措施。然而，治疗时间窗短、出血转化、溶栓灌注率低和神经毒性是限制其应用的主要因素。在这项建议中，我们假设重组Annexin A2蛋白(RA2)将降低再灌注所需的tPA剂量，同时增强溶栓疗效，并减轻脑出血转化。通过这样做，它将延长治疗时间窗口并改善长期结果。我们这一想法的分子基础是膜联蛋白A2通过与tPA和纤溶酶前体纤溶酶原组装纤溶复合体来加速溶解血栓的纤溶酶的产生。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所涉及的机制。在目标1中，我们将研究RA2和小剂量tPA联合治疗在大鼠局灶性脑梗塞后4小时的溶栓效果，用MR血管造影确定再通和再闭塞，MRI脑血流图显示脑血流，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。目的2观察RA2加小剂量tPA联合治疗对大鼠局灶性脑梗塞后4小时神经血管基质蛋白降解和血脑屏障漏的影响。将测量脑梗塞、水肿和出血性转化的发展，并将其与基质金属蛋白酶-9活性、细胞外基质成分的蛋白降解和血脑屏障渗漏的时间分布相关联。在目标3中，我们将评估卒中后4小时RA2加小剂量tPA联合治疗的长期结果。老鼠将在4周内接受一系列行为测试。最后处死动物，测量脑组织血管密度和组织修复因子血管内皮生长因子、血管紧张素转换酶1的表达。将低剂量tPA+RA2与单独使用RA2、单独使用低剂量和高剂量tPA以及车用生理盐水对照进行比较。如果成功，这些拟议的实验将为RA2加低剂量tPA如何改善tPA卒中治疗提供新的见解，并最终可能产生一种新的组合方法来优化基于tPA的溶栓卒中治疗。我们认为我们的建议应该与PA-07-233，“重症监护中的多学科转化性研究”(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).