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

在我们成功地表征了抗pf4 /聚阴离子抗体（aPF4/P Abs）的生物物理特性后，我们现在的目标是在这些信息的基础上进一步了解HIT的发病机制。我们在之前的研究中已经表明，临床相关的aPF4/P抗体比临床不相关的抗体对PF4/肝素复合物具有更高的结合亲和力，我们现在的目标是在不同的物理化学环境和突变/重组PF4下广泛研究抗体与PF4/P复合物的相互作用。作为第二种方法，我们希望利用细胞膜模拟系统建立aPF4/P抗体的检测方法，因为临床相关的抗体与细胞膜上的PF4/P复合物的结合比临床不相关的抗体具有更高的亲和力。通过这两种方法，我们旨在建立一种区分临床相关（血小板激活）和非相关aPF4/P抗体的工具，该工具可以进一步开发用于临床应用。单克隆抗体是非常有用的致病性研究工具，但两种可用的模仿人aPF4/P抗体的单克隆抗体，即KKO和5B9，与患者来源的抗体相比，没有很好的表征。利用我们在过去3年中开发的工具，我们的目标是将这两种单克隆抗体与人、患者来源的aPF4/P抗体进行比较，以促进对这些抗体未来在体外和体内研究（动物研究）的解释。在我们的建议的第三部分，我们讨论了HIT的发病机制。我们观察到PF4与血管性血友病因子结合，这开启了aPF4/P抗体干扰ADAMTS13切割血管性血友病因子的可能性，从而导致血栓性微血管病变。根据我们的临床观察，我们假设自身免疫组-3抗体干扰血管性血友病因子的ADAMTS 13切割，因为这些抗体的患者表现出血栓性微血管病变的症状。与这个问题密切相关的是本提案的最后一部分，我们的目标是进一步研究aPF4/P抗体干扰和激活内皮细胞的机制。综上所述，本提案将对HIT的发病机制提供更深入的了解，并可能为解决与HIT相关的最重要的临床问题之一提供基础信息，即提高临床实验室适用于临床相关aPF4/P抗体的检测的特异性。

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.