Biological Significance of Protatmine/Heparin Antibodies

描述(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批准的唯一逆转肝素(H)抗凝的疗法。鱼精蛋白的几种不良反应已被描述，包括过敏反应、心血管衰竭和死亡。有发生鱼精蛋白反应风险的人群包括：接受含有鱼精蛋白的胰岛素制剂(如中性鱼精蛋白Hagedorn、NPH胰岛素)的患者、鱼过敏患者和输精管结扎术男性。我导师的实验室在小鼠模型中表明，PrT与肝素相互作用，形成免疫原性多分子复合体。基于这些发现，我在我的研究奖学金期间(从2011年7月开始)进行了研究，以展示人类体内PRT/H复合体的抗体。为此，我从最近的一项多中心前瞻性研究中调查了500名患者，调查了心脏手术后血小板因子4(Pf4)/H抗体的发生率。 手术(点击5801)。在IRB的批准下，我确定PRT/H抗体在心脏手术后常见(CPB后30天发生率为29%)，并与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激活。基于这些观察，我假设PRT/H抗体在循环抗原(细胞结合的PRT或PRT/H复合体)存在的情况下是致病的。我将在以下两个目标中验证这一假设：目标1：证明PRT/H抗体的细胞致病性。在初步研究中，我证明了PRT/H抗体激活单核细胞表达促凝血活性，并与NPH胰岛素中的PRT发生交叉反应。在这个目标中，我将检验PRT/H抗体通过抗原和抗体介导的作用发挥不同生物学效应的假设。具体地说，我将研究PRT/H抗体对葡萄糖摄取、胰岛素受体激活和细胞促凝活性的功能影响。此外，我还将研究PRT/H抗体与其他鱼精蛋白样物质(组蛋白)的交叉反应。目的2：制备小鼠抗PRT/H的单抗。由于人的标本有限，我将制备一种小鼠抗PRT/H的单抗。该单抗将成为未来体外和体内研究的重要工具。如果成功，这些研究将为未来有关PRT/H抗体在需要接触鱼精蛋白的易感患者群体中的临床影响和致病潜力的研究提供信息，例如胰岛素依赖型糖尿病患者、外周动脉疾病患者或重复心脏手术的患者。

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.