Biological Significance of Protatmine/Heparin Antibodies

描述（由申请人提供）：鱼精蛋白/肝素抗体的生物学意义鱼精蛋白（PRT）是目前FDA批准的唯一一种逆转肝素（H）抗凝的疗法。使用鱼精蛋白时出现了几种不良反应，包括过敏反应、心血管性虚脱和死亡。有发生鱼精蛋白反应风险的人群包括：接受含鱼精蛋白胰岛素制剂（如中性鱼精蛋白哈格多恩、NPH胰岛素）的患者、鱼类过敏患者和输精管切除男性。我导师的实验室已经表明PRT与肝素相互作用，在小鼠模型中形成免疫原性多分子复合物。基于这些发现，我在研究期间（从2011年7月开始）进行了研究，以证明人类中PRT/H复合物的抗体。为此，我调查了最近一项多中心前瞻性研究中的500例患者，该研究调查了心脏起搏后血小板因子4（PF 4）/H抗体的发生率。 手术（HIT 5801）。经IRB批准，我确定PRT/H抗体通常发生在心脏手术后（CPB后30天发生率为29%），并且与HIT抗体具有几个基本特性，包括：肝素依赖性、抗原特异性和血小板活化。基于这些观察结果，我假设PRT/H抗体在循环抗原（细胞结合PRT或PRT/H复合物）存在下具有致病性。我将在以下两个目标中检验这一假设：目标1：证明PRT/H抗体的细胞致病性。在初步研究中，我证明，PRT/H抗体激活单核细胞表达促凝血活性和抗体交叉反应与PRT在NPH胰岛素。在这个目标中，我将测试的假设，PRT/H抗体发挥不同的生物学效应，通过抗原和抗体介导的影响。具体来说，我将检查PRT/H抗体对葡萄糖摄取，胰岛素受体激活和细胞促凝血活性的功能性影响。此外，我将研究PRT/H抗体与其他鱼精蛋白样物质（组蛋白）的交叉反应性。目的2：制备抗PRT/H的鼠源性单克隆抗体。由于人样本的可用性有限，我将制备鼠单克隆PRT/H抗体。这种单克隆抗体将是未来体外和体内研究的重要工具。如果成功，这些研究将为未来研究PRT/H抗体在需要鱼精蛋白暴露的易感患者人群（如胰岛素依赖型糖尿病、外周动脉疾病或重复心脏手术患者）中的临床影响和致病潜力提供信息。

DESCRIPTION (provided by applicant): Biological Significance of Protamine/Heparin Antibodies Protamine (PRT) is currently the only FDA approved therapy for reversing heparin (H) anticoagulation. Several adverse reactions have been described with protamine use including anaphylaxis, cardiovascular collapse, and death. Populations at risk for developing a protamine reaction include: patients receiving protamine-containing insulin preparations (such as Neutral Protamine Hagedorn, NPH insulin), patients with fish allergies, and vasectomized men. My mentor's laboratory has shown that PRT interacts with heparin to form immunogenic multi- molecular complexes in a murine model. Based on these findings, I undertook studies during my research fellowship (starting July 2011) to demonstrate antibodies to PRT/H complexes in humans. To do so, I investigated 500 patients from a recent multicenter, prospective study investigating the incidence of platelet factor 4 (PF4)/H antibodies after cardiac surgery (HIT 5801). With IRB approval, I determined that PRT/H antibodies occur commonly after cardiac surgery (29% incidence 30 days post-CPB) and share several essential properties with HIT antibodies including: heparin-dependence, antigen-specificity, and platelet activation. Based on these observations, I hypothesize that PRT/H antibodies are pathogenic in the presence of circulating antigen (cell bound PRT or PRT/H complexes). I will test this hypothesis in the following two Aims: Aim 1: Demonstrate the cellular pathogenicity of PRT/H antibodies. In preliminary studies, I demonstrate that PRT/H antibodies activate monocytes to express procoagulant activity and antibodies cross-react with PRT in NPH insulin. In this aim, I will test the hypothesis that PRT/H antibodies exert diverse biological effects through antigen and antibody-mediated effects. Specifically, I will examine the functional effects of PRT/H antibodies on glucose uptake, insulin receptor activation and cellular procoagulant activity. Additionally, I will investigate the cross-reactivity of PRT/H antibodies with other protamine like substances (histones). Aim 2: Generate murine monoclonal antibodies to PRT/H. Due to limited availability of human specimens, I will generate a murine monoclonal PRT/H antibody. This monoclonal antibody will be an important tool for future in vitro and in vivo studies. If successful, these studies will inform future investigations on the clinical impact and pathogenic potential of PRT/H antibodies in susceptible patient populations requiring protamine exposure, such as patients with insulin dependent diabetes, peripheral arterial disease or repeat cardiac surgery.