DataSheet4_Plasma enzymatic activity, proteomics and peptidomics in COVID-19-induced sepsis: A novel approach for the analysis of hemostasis.PDF

Introduction: Infection by SARS-CoV-2 and subsequent COVID-19 can cause viral sepsis. We investigated plasma protease activity patterns in COVID-19-induced sepsis with bacterial superinfection, as well as plasma proteomics and peptidomics in order to assess the possible implications of enhanced proteolysis on major protein systems (e.g., coagulation).
Methods: Patients (=4) admitted to the intensive care units (ICUs) at the University of California, San Diego (UCSD) Medical Center with confirmed positive test for COVID-19 by real-time reverse transcription polymerase chain reaction (RT-PCR) were enrolled in a study approved by the UCSD Institutional Review Board (IRB# 190699, Protocol #20-0006). Informed consent was obtained for the collection of blood samples and de-identified use of the data. Blood samples were collected at multiple time points and analyzed to quantify a) the circulating proteome and peptidome by mass spectrometry; b) the aminopeptidase activity in plasma; and c) the endopeptidase activity in plasma using fluorogenic substrates that are cleaved by trypsin-like endopeptidases, specific clotting factors and plasmin. The one patient who died was diagnosed with bacterial superinfection on day 7 after beginning of the study.
Results: Spikes in protease activity (factor VII, trypsin-like activity), and corresponding increases in the intensity of peptides derived by hydrolysis of plasma proteins, especially of fibrinogen degradation products and downregulation of endogenous protease inhibitors were detected on day 7 for the patient who died. The activity of the analyzed proteases was stable in survivors.
Discussion: The combination of multiomics and enzymatic activity quantification enabled to i) hypothesize that elevated proteolysis occurs in COVID-19-induced septic shock with bacterial superinfection, and ii) provide additional insight into malfunctioning protease-mediated systems, such as hemostasis.

引言：严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）感染及其引发的新型冠状病毒肺炎（COVID-19）可诱发病毒性脓毒症。本研究针对合并细菌双重感染的新冠诱导脓毒症患者，分析其血浆蛋白酶活性谱，并结合血浆蛋白质组学与肽组学数据，以评估蛋白水解增强对凝血系统等主要蛋白系统的潜在影响。
方法：本研究经加州大学圣地亚哥分校（University of California, San Diego, UCSD）医学中心伦理审查委员会批准（伦理编号IRB# 190699，方案编号20-0006），纳入该中心重症监护病房（Intensive Care Units, ICUs）收治的、经实时逆转录聚合酶链式反应（real-time reverse transcription polymerase chain reaction, RT-PCR）确认新冠病毒核酸阳性的4例患者。所有受试者均签署知情同意书，同意采集血液样本并进行数据的去标识化使用。研究采集多个时间点的血液样本，开展以下分析：a) 采用质谱技术定量检测循环蛋白质组与肽组；b) 检测血浆氨基肽酶活性；c) 利用荧光底物检测血浆内肽酶活性，该底物可被类胰蛋白酶内肽酶、特异性凝血因子及纤溶酶切割。其中1例死亡患者在研究启动后第7天被确诊为细菌双重感染。
结果：该死亡患者在研究启动后第7天，检测到蛋白酶活性（凝血因子Ⅶ、类胰蛋白酶活性）出现峰值，血浆蛋白水解产生的肽段信号强度显著升高（尤以纤维蛋白原降解产物为著），同时内源性蛋白酶抑制剂表达下调；而存活患者的目标蛋白酶活性维持稳定。
讨论：本研究结合多组学与酶活性定量分析，可实现两点研究进展：其一，提出合并细菌双重感染的新冠诱导脓毒性休克患者体内存在蛋白水解增强现象的假说；其二，为凝血功能异常等蛋白酶介导的系统功能障碍提供了额外的研究见解。