Characterization of Platelet factor 4-Polyanion complexes and corresponding pathogenic antibodies

在我们成功地表征了抗PF 4/聚阴离子抗体（aPF 4/P Abs）的生物物理特性后，我们现在的目标是在此基础上进一步了解HIT的发病机制。我们在之前的提议中已经表明，临床相关的aPF 4/P Ab与PF 4/肝素复合物的结合亲和力高于临床不相关的Ab，我们现在的目标是使用不同的物理化学环境和突变/重组的PF 4广泛研究Ab与PF 4/P复合物的相互作用。作为第二种方法，我们希望使用细胞膜模拟系统建立aPF 4/P Ab的检测方法，因为临床相关Ab与细胞膜上的PF 4/P复合物的结合亲和力比临床非相关抗体高得多。我们的目标是建立一个工具来区分临床相关的（血小板活化）和非相关的aPF 4/P Abs，这可以进一步开发用于临床应用。单克隆抗体是非常有用的工具，用于致病性研究，但两个可用的单克隆抗体模仿人aPF 4/P Abs，即KKO和5 B 9没有得到很好的表征相比，患者来源的抗体。与我们在过去3年中开发的工具，我们的目标是表征这两个单克隆抗体相比，人类，患者来源的aPF 4/P抗体，以促进解释未来的体外和体内研究（动物研究）与这些antibodies.In第三部分，我们的建议，我们解决HIT的发病机制。我们建立在PF 4与血管性血友病因子结合的观察结果的基础上，这开启了aPF 4/P Ab干扰ADAMTS 13对血管性血友病因子的切割从而引起血栓性微血管病的可能性。基于我们的临床观察，我们假设自身免疫性第3组抗体干扰血管性血友病因子的ADAMTS 13裂解，因为具有这些抗体的患者显示血栓性微血管病的症状。与此密切相关的是本研究的最后一部分，我们的目标是进一步研究aPF 4/P Abs干扰和激活内皮细胞的机制，从而加深对HIT发病机制的理解，并为解决HIT相关的最重要的临床问题之一提供基础信息。即，提高临床实验室中适用于临床相关aPF 4/P Ab的检测的特异性。

After we have successfully characterized biophysical properties of anti-PF4/polyanion antibodies (aPF4/P Abs), which induce the adverse drug effect heparin-induced thrombocytopenia (HIT), we now aim to build on this information to further understand the pathogenesis of HIT. We have shown in our previous proposal that clinically relevant aPF4/P Abs have a higher binding affinity to PF4/heparin complexes than clinically non-relevant Abs, we now aim to widely investigate the interaction of Abs to PF4/P complexes using different physicochemical environments and mutated/recombinant PF4s. As second approach, we want to establish a detection method for aPF4/P Abs using a cell membrane mimicking system because the clinically relevant Abs bind with much higher avidity to PF4/P complexes on cell membranes than clinically non-relevant antibodies. With both approaches we aim to establish a tool to differentiate clinically relevant (platelet activating) and non-relevant aPF4/P Abs, which can be further developed for clinical application.Monoclonal antibodies are very helpful tool for pathogenicity studies, but the two available monoclonal antibodies mimicking human aPF4/P Abs, namely KKO and 5B9 are not well-characterized in comparison to patient-derived antibodies. With the tools we have developed during the last 3 years, we aim to characterize both monoclonal Abs in comparison to human, patient-derived aPF4/P Abs to facilitate interpretation of future in vitro and in vivo studies (animal studies) with these antibodies.In the third part of our proposal we address the pathogenesis of HIT. We build on the observation that PF4 binds to von Willebrand factor which opens the possibility that aPF4/P Abs interfere with von Willebrand factor cleavage by ADAMTS13 causing thrombotic microangiopathy. Based on our clinical observations, we assume that autoimmune group-3 antibodies interfere with ADAMTS 13 cleavage of von Willebrand factor, as patients with these antibodies show symptoms of thrombotic microangiopathy. Closely linked to this question is the final part of this proposal in which we aim to further investigate the mechanisms by which aPF4/P Abs interfere and activate endothelial cells.Taken together, this proposal will provide deeper understanding on the pathogenesis of HIT, and may provide the basic information to solve one of the most important clinical issues associated with HIT, i.e. improving the specificity of tests applicable in the clinical laboratory for clinically relevant aPF4/P Abs.