Recombinant annexin A2 plus tPA for combination stroke therapy

描述（由申请方提供）：重组tPA静脉给药仍然是唯一获得FDA批准的、经证实最有益的卒中紧急治疗干预措施。然而，治疗时间窗短、出血性转化、溶栓灌注率差和神经毒性构成了应用的主要限制。在这个提议中，我们假设重组膜联蛋白A2蛋白（rA 2）将降低再灌注所需的tPA剂量，同时提高溶栓疗效，并减弱脑内出血转化。通过这样做，它将延长治疗时间窗并改善长期结果。我们的想法的分子基础是膜联蛋白A2通过与tPA和纤溶酶前体纤溶酶原组装纤溶复合物来加速凝块溶解纤溶酶的产生。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治疗中涉及的机制。 在目标1中，我们将研究大鼠局灶性栓塞性卒中后4小时rA 2加低剂量tPA联合治疗的溶栓疗效，采用MR血管造影术确定再通和再闭塞，MRI CBF绘制脑血流量; MRI测量脑血容量（CBV）、微血管容积（MVV）和血管尺寸指数（VSI），以量化血管对溶栓药物和血管扩张剂的反应。在目标2中，我们将研究rA 2加低剂量tPA联合治疗对大鼠局灶性栓塞性卒中后4小时治疗的神经血管基质蛋白水解和BBB渗漏的影响。将测量脑梗塞、水肿和出血性转化的发展，并将其与MMP-9活性、细胞外基质组分的蛋白水解降解和BBB渗漏的时间曲线相关联。在目标3中，我们将评估在中风后4小时治疗的rA 2加低剂量tPA组合的长期结果。将对大鼠进行为期4周的一系列行为测试。最后，取脑进行H&E病灶大小测量，检测脑血管密度和组织恢复因子VEGF、Ang-1表达。将低剂量tPA加rA 2与单独的rA 2、单独的低剂量和高剂量tPA以及溶剂盐水对照进行比较。 如果成功，这些拟议的实验应该提供新的见解rA 2加低剂量tPA如何改善tPA中风治疗，并可能最终产生一种新的组合方法，以优化基于tPA的溶栓中风治疗。我们认为我们的建议应该与PA-07-233“危重病护理中的多学科转化研究”（R 01）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).