Covid-19 Spike Proteins Form Antigenic Complexes with Platelet Factor 4 that Trigger Production of Pathogenic Antibodies: A New Mechanism of Autoimmunity Thrombocytopenia

截至2021年3月，世卫组织报告自疫情开始以来全球约有11588.7万例感染病例和257.4万例死亡。71.4%的新冠肺炎患者死亡时凝血功能异常。最近，在Covid-19患者中检测到不同类型的致病性抗体Ab，这些抗体通过FcγRIIa激活血小板并导致最严重的凝血。然而，触发机体产生这些抗体的机制尚未阐明。最近，我们利用纳米生物物理学方法发现了由抗血小板因子4（PF 4）抗体（aPF 4 Abs）介导的自身免疫的新机制（Nguyen et al，NCOMMS，2017）。这些aPF 4 Ab对PF 4具有高结合亲和力，因为它们聚集PF 4分子并形成PF 4/抗体复合物，该复合物暴露肝素（H）诱导的血小板减少症（HIT）抗体的结合表位。在这个项目中，我们的目标是转让我们的技术，以实现Covid-19刺突蛋白（SP）在Covid-19患者致病性抗体产生中的作用。我们假设SP具有与aPF 4 Ab相似的聚集PF 4分子的能力，形成PF 4/SP复合物，促进免疫细胞产生不同类型的致病抗体，这些抗体通过FcγRIIa激活血小板和其他血细胞。为了证明这一机制，我们首先鉴定SP是否对PF 4具有高结合亲和力，并且也对PF 4/通过评价相互作用的结合力和动力学性质的肝素复合物。高结合亲和力表明SP可以使PF 4和PF 4/H复合物成簇。我们接下来测试所得的PF 4/SP和PF 4/H/SP复合物是否允许结合不同类型的血小板活化抗体。作为模型，我们将使用充分表征的单克隆HIT抗体（KKO）、抗PF 4抗体和肾抗体。如果这些结合发生，我们怀疑免疫细胞可能会在Covid-19患者体内产生高浓度的这些抗体。我们将分离并测定这些抗体的浓度，包括aPF 4/SP Abs、aPF 4/H/S Abs、HIT Abs、aPF 4 Abs和肾脏Abs，并检测它们是否含有FcγRIIa。如果这些抗体在血清中的浓度很高，并且它们激活血小板，我们提出的机制就被证明了。最后，我们的目的是确定肝素，可以降低SP的ACE 2，PF 4，和PF 4/H复合物的结合亲和力。正如我们先前发现的不同长度的合成肝素比动物来源的肝素具有更高的活性，我们将应用纳米生物物理方法来确定潜在的肝素，可以降低SP的反应性。我们假设，补丁的正电荷的病毒可以被封锁的负电荷的肝素大小相等。综上所述，这一提议将提供Covid-19 SP在聚集PF 4中的新机制，导致Covid-19患者产生侵袭性病原性抗体。此外，我们可能能够确定肝素，降低SP的反应性。

Until March 2021, the WHO reported around 115.887 million infected cases and 2.574 million deaths globally since the start of the pandemic. A 71.4% of the deaths in Covid-19 patients had abnormal coagulation profiles. Recently, different types of pathogenic antibodies Abs that activate platelets via FcγRIIa and caused the worst clotting, have been detected in Covid-19 patients. However, the mechanism of what triggering the body to produce these antibodies has not yet been clarified.Recently, we utilized nanobiophysical methods to discover a new mechanism of autoimmunity mediated by anti-platelet factor 4 (PF4) antibodies (aPF4 Abs) (Nguyen et al, NCOMMS, 2017). These aPF4 Abs have a high binding affinity to PF4 as they cluster PF4 molecules and form PF4/antibody complexes that expose binding epitope for heparin (H)-induced thrombocytopenia (HIT) antibodies. In this project, we aim to transfer our technologies to realize the role of Covid-19 spike proteins (SP) in the production of pathogenic antibodies in Covid-19 patients. We hypothesize that SP have a capacity in clustering PF4 molecules similar to the aPF4 Abs, forming PF4/SP complexes that promote immune cells to produce different types of pathogenic antibodies, which activate platelets and other blood cells via FcγRIIa.To prove this mechanism, we first identify if SP have a high binding affinity to PF4 and also to PF4/Heparin complexes via evaluation of binding forces and kinetic properties of the interactions. High binding affinity indicates that SP can cluster PF4 and PF4/H complexes.We next test if the resulting PF4/SP and PF4/H/SP complexes allow binding different types of platelet-activating antibodies. As a model, we will use well-characterized monoclonal HIT Abs (the KKO), the anti-PF4 Abs, and the kidney Abs. If these bindings occur, we suspect that immune cells may produce a high concentration of these antibodies in Covid-19 patients. We will isolate and determine the concentration of these antibodies including aPF4/SP Abs, aPF4/H/S Abs, HIT Abs, aPF4 Abs, and kidney Abs in Covid-19 patients, and test if they contain FcγRIIa. If concentrations of these Abs are high in sera and they activate platelets, our proposed mechanism is proven. Finally, we aim to identify the heparins that can reduce the binding affinity of SP to the ACE2, PF4, and PF4/H complexes. As we found previously that the synthetic heparins of different lengths exhibited higher activity than the animal-derived heparins, we will apply nanobiophysical methods to identify potential heparins that can reduce the reactivity of SP. We hypothesize that the patches of the positive charge of the virus can be blocked by the negatively charged heparin of equal size. Taken together, this proposal will provide a new mechanism of Covid-19 SP in clustering PF4 that leads to the production of aggressive pathogenic antibodies in Covid-19 patients. Furthermore, we may be able to identify heparins that reduce the reactivity of SP.