Ma Xiaoying, Liang Jady, Zhu Guangheng, Bhoria Preeti, Shoara Aron A, MacKeigan Daniel T, Khoury Christopher J, Slavkovic Sladjana, Lin Lisha, Karakas Danielle, Chen Ziyan, Prifti Viktor, Liu Zhenze, Shen Chuanbin, Li Yuchong, Zhang Cheng, Dou Jiayu, Rousseau Zack, Zhang Jiamin, Ni Tiffany, Lei Xi, Chen Pingguo, Wu Xiaoyu, Shaykhalishahi Hamed, Mubareka Samira, Connelly Kim A, Zhang Haibo, Rotstein Ori, Ni Heyu
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, ON, Canada.
Research (Wash D C). 2023 Apr 24;6:0124. doi: 10.34133/research.0124. eCollection 2023.
The COVID-19 pandemic caused by SARS-CoV-2 virus is an ongoing global health burden. Severe cases of COVID-19 and the rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are both associated with thrombosis and thrombocytopenia; however, the underlying mechanisms remain inadequately understood. Both infection and vaccination utilize the spike protein receptor-binding domain (RBD) of SARS-CoV-2. We found that intravenous injection of recombinant RBD caused significant platelet clearance in mice. Further investigation revealed the RBD could bind platelets, cause platelet activation, and potentiate platelet aggregation, which was exacerbated in the Delta and Kappa variants. The RBD-platelet interaction was partially dependent on the β3 integrin as binding was significantly reduced in β3 mice. Furthermore, RBD binding to human and mouse platelets was significantly reduced with related αIIbβ3 antagonists and mutation of the RGD (arginine-glycine-aspartate) integrin binding motif to RGE (arginine-glycine-glutamate). We developed anti-RBD polyclonal and several monoclonal antibodies (mAbs) and identified 4F2 and 4H12 for their potent dual inhibition of RBD-induced platelet activation, aggregation, and clearance in vivo, and SARS-CoV-2 infection and replication in Vero E6 cells. Our data show that the RBD can bind platelets partially though αIIbβ3 and induce platelet activation and clearance, which may contribute to thrombosis and thrombocytopenia observed in COVID-19 and VITT. Our newly developed mAbs 4F2 and 4H12 have potential not only for diagnosis of SARS-CoV-2 virus antigen but also importantly for therapy against COVID-19.
由严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒引起的2019冠状病毒病（COVID-19）大流行是一项持续存在的全球健康负担。COVID-19的重症病例以及罕见的COVID-19疫苗诱导的血栓性血小板减少症（VITT）病例均与血栓形成和血小板减少有关；然而，其潜在机制仍未得到充分了解。感染和疫苗接种均利用SARS-CoV-2的刺突蛋白受体结合域（RBD）。我们发现，静脉注射重组RBD可导致小鼠体内血小板显著清除。进一步研究表明，RBD可结合血小板，引起血小板活化，并增强血小板聚集，在Delta和Kappa变体中这种情况会加剧。RBD与血小板的相互作用部分依赖于β3整合素，因为在β3基因敲除小鼠中结合显著减少。此外，使用相关的αIIbβ3拮抗剂以及将RGD（精氨酸-甘氨酸-天冬氨酸）整合素结合基序突变为RGE（精氨酸-甘氨酸-谷氨酸）后，RBD与人及小鼠血小板的结合显著减少。我们制备了抗RBD多克隆抗体和几种单克隆抗体（mAb），并鉴定出4F2和4H12，它们在体内对RBD诱导的血小板活化、聚集和清除以及在Vero E6细胞中的SARS-CoV-2感染和复制具有强大的双重抑制作用。我们的数据表明，RBD可部分通过αIIbβ3结合血小板并诱导血小板活化和清除，这可能导致COVID-19和VITT中观察到的血栓形成和血小板减少。我们新开发的单克隆抗体4F2和4H12不仅具有诊断SARS-CoV-2病毒抗原的潜力，而且对于COVID-19的治疗也具有重要意义。
