В работе исследователей из Массачуссетса использованы "гуманизированные" мыши (с человеческими АСЕ2), для того, чтобы изучить патологию тромбообразования, как результат воздействия коронавируса SARS-CoV-2, с помощью протеомного анализа и масс-спектрометрии.
При инфицировании таких мышей у них так же, как и у больных тяжелым ковидом людей, развивалась прогрессирующая лимфогистиоцитарная интерстициальная пневмония с микротромбами в легких и почках.
Авторы обнаружили значительные изменения примерно в 1200 белках тромбоцитов после инфекции, причем начиная уже через 2 дня после заражения мышей. Анализ показал, что инфекция SARS-CoV-2 активирует взаимодействия системы свертывания крови и комплемента (F2, F12, CFH, CD55 / CD59), белки активации-адгезии-дегрануляции тромбоцитов (PF4, SELP, PECAM1, HRG, PLG, vWF) и выработку хемокинов (CCL8, CXCL5, CXCL12). На 4 день после инфекции в тромбоцитах были гиперактивированы сигнальные пути рецептора распознавания паттернов (RIG-I / MDA5, CASP8, MAPK3) и сигнальные пути интерферона (IFIT1 / IFIT3, STAT1). При этом, у больных мышей шипиковый белок обнаруживался только внутри клеток легких, но не в тромбоцитах.
Так что в работе показано, что вирус "перепрограммирует" циркулирующие тромбоциты, провоцируя их дегрануляцию, чрезмерно стимулируя их взаимодействие с ситемой комплемента и склеивание, начиная уже со 2 дня инфекции, и "про-тромбозные" микроскопические изменения начинаются в тканях раньше, чем они проявляются патологически тяжестью болезни, а интерферон тромбоциты начинают выбрасывать примерно через 4 дня после заражения.
Hospitalized COVID-19 patients frequently develop coagulation abnormalities with a high risk22 (~10-40%) of thromboembolism.1Elevated fibrin-degradation products are predictorsof mortality from COVID-19,2but therapeutic anticoagulation with heparin has achieved inconsistent survival benefit sin clinical trials.3,4Hence, there remains urgent need for molecular insight into COVID- 19-associated coagulopathy (CAC), which shares some features with disseminated intravascular coagulopathy. During CAC, human platelets are activated and participate in procoagulatory- inflammatory responses to COVID‐19.6-8RNA-Seq has revealed what appears to be both direct and indirect effects (e.g. mediators, aberrant antibodies) of SARS-CoV-2 infection on the platelet transcriptome in patients. Mouse models are essential tools for drug development. While infected hACE2 mice present with microthrombosis, detailed platelet characterizations remain lacking.Here, we have phenotyped the platelet proteomes of SARS-CoV-2-infected humanized mice under standardized conditions and defined time points to provide an instrumental resource for future studies. As platelet-fibrin thrombi characterize multi-organ thrombosis of lethal COVID-19in humans, we examined mouse lungs and kidneys at two time points (2dpiand 4dpi). Histologic findings included multifocal mild-to-moderate mononuclear cell infiltrates and low numbers of neutrophils in peribronchiolar, perivascular, interstitial, and alveolar spaces . SARS-CoV-2 spike protein was observed in the alveolar epithelium. Although there were no occlusive thrombi, we observed sporadic enhanced aggregations of CD61+platelets in lung capillaries and kidney interstitial/glomerular capillaries . Lung interstitial capillaries within areas of viral replication/assembly sporadically contained platelet aggregates .These findings suggest platelet activation and aggregation is a feature of SARS-CoV-2 infection of K18-hACE2 mice already occurring before clinical decline. The analysis at 2dpi revealed a significant increase in SELP, PECAM-1, PLG, vWF16as well as thrombinexpression , supporting both the activation and degranulationof platelets during SARS-CoV-2 infection. Thrombin causes platelets to change shape, aggregate, and secrete prothrombotic granule contents. Elevated platelet thrombin might be due to circulating thrombin binding to platelet PAR3, which then activates platelet PAR4 during infection,although this needs further investigation. Similarly, an increase in coagulation factor F12 at 2dpi suggests plasma F12 may bind platelet sto accelerate activation (contact pathway),and participate in CAC. Furthermore, reduced F5at 2dpi could indicate F5 release to the plasma by activated platelets.On the other hand, we also observed increased anti-coagulation proteins (TFPI, PROC, PROS1, THBD) , suggesting both pro-coagulantand anti-80coagulant factors were prominently dysregulated during early SARS-CoV-2 infection. Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multi-domain structure, allowing interaction with many ligands including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate.HRGwas significantly increased at 2dpi and 4dpi . 85HRGexerts broad antiviral activities and might help to explain platelet-mediated antiviral 86responsesduringSARS-CoV-2infection. In contrast, HRG may also have deleterious effects. 87We speculate that HRG-coated, activated platelets could adhere to inflamed endothelium via heparan sulfate, which would then block antithrombin-III binding and FXa inhibition, and could ultimately promote thromboembolism/ microthrombi in arterioles and venules Platelet activation during thrombosis is closely associated with complement and contact system activation,antibody attack, and in turn inflammation. Interestingly, complement regulatory proteins (CD55, CD59, C1R, CFH,CFI)and membrane-attack complex component 8(C8G) were significantly upregulated at 2dpi . Similar to atypical hemolytic uremic syndrome, factor H (CFH) activity on platelets at 2dpi could be associated with platelet aggregation and activation. The significant increase in platelet CD55 and CD59 (GPI-anchored membrane proteins),and C1q-associated protease (C1R), support both complement system activation and plateletprotectionfrom complement-mediated destruction during SARS-CoV-2 infection. Of note, significantly upregulated proteins at 4dpi were enriched in pathways typically engaged in the response to infection with viruses (Influenza A) and other intracellular pathogens (e.g., Salmonella). These included NOD-like receptor signaling (CASP8, GBP3, GBP2, MAPK3) , and Toll-like and RIG-I-like receptor signaling pathways (DDX58/RIG-I, IFIH1/MDA5, DHX58, 104IRAK4, RAC1, MAPK3)(Figure 2K, 2L). In line with this, we observed dominant interferon-induced 105proteins (IFIT1, IFITM3) and STAT1 in platelets at 4dpi, suggesting a platelet immune response . In addition to these pathways, certain chemokines (PF4, CCL8) and TNFα signaling components (RIPK3, CASP8) were strongly upregulated at both 2dpi and 4dpi compared to uninfected controls.PF4/CXCL4 is of particular interest as the most abundant platelet kinocidin. It is released from α-granules of activated platelets . Importantly, platelets from COVID‐19 patients have been shown to release δ/α-granule cargo into the blood. PF4 has also been implicated in vaccine-induced thrombotic responses. In addition, CXCL12, CXCL5, and CCL8 were likewise elevated . Тhese chemokines recruit monocytes/ macrophages to infection sites and promote platelet-leukocyte aggregates. A protein-protein interaction network of significantly upregulated proteins at 2dpi and 4dpi was retrieved from the STRING database, which revealed modules with multiple sources of biological crosstalk .For instance, vWF has been reported to play a critical role in COVID-19 pulmonary microvascular occlusion. In our interactome, we noted high interconnectivity between vWF, PLG, PF4, ITGB3 and MAPK3, as well as between PF4,CXCL12 and CXCL5. Dysregulation of vWF, F2,and PF4may serve as central nodes in enhancing virus-induced cytopathologysimilar to RSV by interacting with proteins governing platelet activation and degranulation, complement activation, coagulation, ECM-receptor interaction, and other key pathways dysregulated in COVID-19.Of note, proteins upregulated at 2dpi were more interconnected compared to 4dpi. Within our K18-hACE2 mouse platelet samples, SARS-CoV-2proteins, ACE2, and TMPRSS2127were not detectable by mass spectrometry .In human platelets, SARS-CoV- 2 uptake seems infrequent, independent of ACE2/TMPRSS2, and can induce cell death pathways
Apoptotic proteins were also upregulated in mouse platelets .In conclusion, our study identified platelet proteome signatures dependent on SARS-CoV-2 infection timing, and validated K18-hACE2 mice as a suitable model for future studies gaging the direct impacts of specific platelet components on COVID-19 progression.