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，世界卫生组织报告称，自疫情开始以来，全球约有11588.7万例感染病例，257.4万人死亡。71.4%的新冠肺炎死亡患者存在凝血功能异常。最近，在Covid-19患者中检测到不同类型的致病抗体，这些抗体通过FcγRIIa激活血小板并引起最严重的凝血。然而，触发人体产生这些抗体的机制尚不清楚。最近，我们利用纳米生物物理方法发现了抗血小板因子4 （PF4）抗体（aPF4 Abs）介导的自身免疫新机制（Nguyen et al， NCOMMS, 2017）。这些aPF4抗体对PF4具有高结合亲和力，因为它们聚集PF4分子并形成PF4/抗体复合物，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，我们的目标是转移我们的技术，以实现Covid-19刺突蛋白（SP）在Covid-19患者产生致病抗体中的作用。我们假设SP具有类似于aPF4抗体聚集PF4分子的能力，形成PF4/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，我们首先通过评估相互作用的结合力和动力学性质来确定SP是否对PF4和PF4/肝素复合物具有高结合亲和力。高结合亲和力表明SP可以聚集PF4和PF4/H复合物。我们接下来测试所得的PF4/SP和PF4/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，我们将使用具有良好特征的单克隆HIT抗体（KKO）、抗pf4抗体和肾抗体。如果发生这些结合，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。我们将在Covid-19患者中分离并测定这些抗体的浓度，包括aPF4/SP抗体、aPF4/H/S抗体、HIT抗体、aPF4抗体和肾抗体，并检测它们是否含有FcγRIIa。如果血清中这些抗体的浓度很高，它们会激活血小板，我们提出的机制就得到了证实。最后，我们的目标是鉴定可以降低SP与ACE2、PF4和PF4/H复合物结合亲和力的肝素。正如我们之前发现的，不同长度的合成肝素比动物源性肝素表现出更高的活性，我们将应用纳米生物物理方法来识别可能降低SP反应性的潜在肝素。我们假设病毒的正电荷斑块可以被大小相等的带负电荷的肝素阻断。综上所述，该建议将提供Covid-19 SP在聚类PF4中导致Covid-19患者产生侵袭性致病抗体的新机制。此外，我们可能能够识别降低SP反应性的肝素。

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.