Biological Significance of Protamine/Heparin Antibodies

我目前是杜克大学四年级的血液学/肿瘤学研究员。在我的T32支持的研究奖学金（5 T32-HL 00572）期间，我专注于描述一类新的肝素依赖性抗体，最初由我的导师的实验室鉴定，鱼精蛋白/肝素抗体。这项工作描述了体外循环手术后鱼精蛋白/肝素抗体的发展，最近发表在血液与“全会文件”的名称。今年，我成功地竞争了F32（1F 32-AI 108118 -01），这使我能够延长一年的博士后培训，为K 08申请生成初步数据。在本申请中，我将建立鱼精蛋白/肝素的生物学意义 抗体的 在我的博士后研究期间，我确定了在体外循环期间单次暴露于鱼精蛋白和肝素后，29%的患者会产生鱼精蛋白/肝素抗体。在我的研究以及其他研究中，发现鱼精蛋白/肝素抗体患者在再次暴露于鱼精蛋白后存在不良临床结局的风险：动脉血栓栓塞事件、血小板减少伴出血和主要不良心血管事件。虽然与发病率相关，但鱼精蛋白/肝素抗体致病的机制尚不清楚。在本K 08提案中，我将确定鱼精蛋白/肝素抗体致病的机制。在初步研究中，我证明鱼精蛋白/肝素抗体诱导促凝血单核细胞组织因子活性，促炎单核细胞IL-8的表达，并直接结合细胞外组蛋白。此外，我证明了成功开发的鼠单克隆抗体鱼精蛋白/肝素，这将有利于我的研究策略中提出的研究。基于这些强有力的初步数据，我将检验鱼精蛋白/肝素抗体在与循环抗原和炎症相关的环境中是致病性的假设。具体而言，在目的1中，我将确定鱼精蛋白/肝素抗体在鱼精蛋白存在下发挥促凝血和促炎作用，导致单核细胞和中性粒细胞活化并释放细胞外组蛋白。在目标2中，我将确定细胞外组蛋白可以作为鱼精蛋白/肝素抗体的抗原并传播细胞损伤。在目标3中，我将使用我最近开发的针对鱼精蛋白/肝素的鼠单克隆抗体来定义鱼精蛋白/肝素抗体的致病特征。在完成所提出的目标，我将学习各种新的体外和体内技术，这将是必不可少的，我作为一个医生科学家的长期成功。 这个K 08奖将使我能够发展必要的学术成功的技能。有了这个奖项，我将实现我的近期目标，了解鱼精蛋白/肝素抗体的生物学意义，也朝着我的长期目标，建立一个独立的研究生涯作为一个医生，科学家在止血和血栓形成的工作。在获奖期间，我不仅将在实验室中掌握一系列新技能，而且还将完成免疫反应的分子和细胞基础的教学课程，并参加各种研讨会，以提高我的学术技能（手稿准备，领导能力，资助写作，科学演示）。杜克大学是一个理想的环境 进行本申请中所述的研究和职业发展计划。它不仅是一个拥有众多资源的成熟研究机构，而且我的部门和部门也致力于我的整体职业发展。我很幸运有一个优秀的导师，Gow Arepally，和一个咨询委员会，他们都致力于我的职业发展，因为我过渡到一个独立的科学生涯。K08 当我过渡到杜克大学的初级教师时，这个奖项将为我提供一个受保护的时间基础。

I am currently a fourth year hematology/oncology fellow at Duke University. During my T32 supported research fellowship (5T32-HL00572), I focused on describing a novel class of heparin-dependent antibodies originally identified by my mentor's laboratory, protamine/heparin antibodies. This work describing the development of protamine/heparin antibodies after cardiopulmonary bypass surgery was recently published in Blood with a "Plenary Paper" designation. This year, I successfully competed for a F32 (1F32-AI108118-01), which allowed me to extend my postdoctoral training for one year to generate the preliminary data for this K08 application. In this application, I will establish the biological significance of protamine/heparin antibodies. During my postdoctoral fellowship, I established that protamine/heparin antibodies develop in 29% of patients after a single exposure to protamine and heparin during cardiopulmonary bypass. In my study, as well as others, patients with protamine/heparin antibodies were found to be at risk for adverse clinical outcomes after re-exposure to protamine: arterial thromboembolic events, thrombocytopenia with bleeding, and major adverse cardiovascular events. Although associated with morbidity, the mechanism of protamine/heparin antibody pathogenicity is unknown. In this K08 proposal, I will determine the mechanisms by which protamine/heparin antibodies are pathogenic. In preliminary studies, I demonstrate that protamine/heparin antibodies induce procoagulant monocyte tissue factor activity, proinflammatory monocyte IL-8 expression, and directly bind to extracellular histones. Additionally, I demonstrate the successful development of a murine monoclonal antibody to protamine/heparin, which will facilitate the studies proposed in my research strategy. Based on this strong preliminary data, I will test the hypothesis that protamine/heparin antibodies are pathogenic in settings associated with circulating antigen and inflammation. Specifically, in Aim 1, I will establish that protamine/heparin antibodies exert procoagulant and proinflammatory effects in the presence of protamine, leading to monocyte and neutrophil activation with release of extracellular histones. In Aim 2, I will establish that extracellular histones can serve as an antigen for protamine/heparin antibodies and propagate cellular injury. In Aim 3, I will define the pathogenic features of protamine/heparin antibodies using a murine monoclonal antibody to protamine/heparin that I have recently developed. In completing the proposed aims, I will learn a variety of new in vitro and in vivo techniques, which will be essential to my long-term success as a physician scientist. This K08 award will allow me to develop the skillset necessary for academic success. With this award, I will achieve my immediate goal of understanding the biological significance of protamine/heparin antibodies and also work towards my long-term goal of establishing an independent research career as a physician-scientist in hemostasis and thrombosis. During the period of the award, I will not only master an array of new skills in the laboratory, but I will alo complete didactic coursework on the molecular and cellular basis of the immune response and participate in a variety of workshops to improve my academic skills (manuscript preparation, leadership skills, grant writing, scientific presentation). Duke University is an ideal environment to pursue the research and career development plan outlined in this application. Not only is it a well-established research institution with a multitude of resources available, but my Department and Division are also committed to my overall career development. I am fortunate to have a superb mentor, Gow Arepally, and an advisory committee who are all dedicated to my career development as I transition to an independent scientific career. This K08 award will provide a foundation of protected time as I transition to junior faculty at Duke University.