Biological Significance of Protamine/Heparin Antibodies

我目前是杜克大学血液学/肿瘤学四年级研究员。在我的T32资助研究奖学金期间(5T32-HL00572)，我专注于描述一种新的肝素依赖抗体，这种抗体最初是由我导师的实验室鉴定的，鱼精蛋白/肝素抗体。这项研究描述了体外循环手术后鱼精蛋白/肝素抗体的发展，最近发表在血液杂志上，并被命名为“全会论文”。今年，我成功竞争了F32(1F32-AI108118-01)，这让我可以将博士后培训延长一年，为这次K08申请生成初步数据。在这项应用中，我将建立鱼精蛋白/肝素的生物学意义 抗体。 在我的博士后研究期间，我发现29%的患者在体外循环期间单次接触鱼精蛋白和肝素后会产生鱼精蛋白/肝素抗体。在我的研究和其他研究中，鱼精蛋白/肝素抗体阳性的患者被发现在再次暴露于鱼精蛋白后面临不良临床结果的风险：动脉血栓栓塞症事件、出血血小板减少症和主要不良心血管事件。虽然与发病率有关，但鱼精蛋白/肝素抗体的致病机制尚不清楚。在这份K08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表达，并直接与细胞外组蛋白结合。此外，我还展示了一种针对鱼精蛋白/肝素的鼠源性单抗的成功研制，这将有助于我的研究策略中提出的研究。基于这一强大的初步数据，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具有致病性的假设。具体地说，在目标1中，我将确定鱼精蛋白/肝素抗体在鱼精蛋白存在的情况下发挥促凝和促炎作用，导致单核细胞和中性粒细胞通过释放细胞外组蛋白而激活。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鼠抗鱼精蛋白/肝素的单抗来定义鱼精蛋白/肝素抗体的致病特性。在完成拟议的目标时，我将学习各种新的体外和体内技术，这将是我作为一名内科科学家取得长期成功所必需的。 这个K08奖项将使我能够培养取得学术成功所必需的技能。有了这个奖项，我将实现我的眼前目标，即了解鱼精蛋白/肝素抗体的生物学意义，并朝着我的长期目标努力，即作为止血和血栓形成方面的内科科学家建立独立的研究生涯。在获奖期间，我不仅将在实验室掌握一系列新技能，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、领导技能、拨款撰写、科学陈述)。杜克大学是理想的环境 执行本申请表中概述的研究和职业发展计划。这不仅是因为它是一个久负盛名的研究机构，拥有大量可用的资源，而且我的部门和部门也致力于我的整体职业发展。我很幸运有一位出色的导师，Gow Aepally，和一个顾问委员会，他们都致力于我的职业发展，因为我正在过渡到一个独立的科学职业生涯。这个K08 当我过渡到杜克大学的初级教师时，该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.