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Representative fluorescence micrographs of purified fluorescent (Alexa Fluor™488) fibrinogen (note no ThT added) with added thrombin to form extensive fibrin clots. A) Fluorescent fibrinogen with thrombin; B) fluorescent fibrinogen with added spike protein (final exposure concentration 1 ng.mL -1 ) and thrombin.
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Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) -induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S...
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... was found that the final exposure concentrations of 1 ng.mL -1 was sufficient and used for the rest of the study (see supplementary raw data for other exposure concentrations micrographs). Figure 3A and B show representative micrographs of purified fluorescent (Alexa Fluor™488) fibrinogen with added thrombin and after exposure to 1 ng.mL -1 spike protein. A denser fibrin clot formed in the presence of spike protein ( Figure 3B). ...
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... 3A and B show representative micrographs of purified fluorescent (Alexa Fluor™488) fibrinogen with added thrombin and after exposure to 1 ng.mL -1 spike protein. A denser fibrin clot formed in the presence of spike protein ( Figure 3B). In PPP, with and without thrombin, the green fluorescent ThT signal indicates areas of amyloid deposit formation. ...
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... addition of thrombin causes purified (Alexa Fluor™488) fibrinogen to polymerize into fibrin networks. Typically, these networks are netlike ( Figure 3A). In the presence of spike protein, the structure changed to form dense clot deposits ( Figure 3B). ...
Similar publications
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) -induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S...
Citations
... For example, Gaebler et al. identified SARS-CoV-2 RNA and protein in 7 of 14 intestinal tissue samples obtained from asymptomatic COVID-19 patients with negative nasal-swab PCR at an average of 4 months after acute disease [58]. The SARS-CoV-2 S antigen S1 itself appears capable of directly interacting with platelets and fibrinogen to drive blood hypercoagulation [59]. This suggests that further studies of convalescent COVID-19 saliva and plasma would benefit from the measurement of SARS-CoV-2 RNA and spike antigen in addition to inflammatory and proteomic signatures. ...
A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal versus pathologic convalescence process have not been well-delineated. Here, we characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 20 and 90 days after initial clinical symptoms resolved. Convalescent subjects showed robust total IgA and IgG responses and positive antibody correlations in saliva and plasma samples. Shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells, such as neutrophil markers (e.g., myeloperoxidase), and clotting factors in plasma (e.g., fibrinogen), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered myeloid-derived pathways that correlated positively with SARS-CoV-2 IgA levels. Beyond plasma, our study positions saliva as a viable fluid to monitor normal and aberrant immune responses including vascular, inflammatory, and coagulation-related sequelae.
... Неврологические нарушения при этой патологии очевидно возникают как в результате прямого цитотоксического действия вируса, так и митохондриальной дисфункции и тромботических нарушений [4]. Тем не менее, у выживших больных основные нарушения центральной нервной системы очевидно будут связаны с последствиями тромбоза мелких сосудов коры головного и мозга и амилоидозом головного мозга и периферической нервной системы, поскольку быстрое образование амилоида, как оказалось, является первичной причиной тромбообразования при COVID-19 [9]. ...
... Амилоид как контактная поверхность в процессе образования тромба В пока единственной работе обнаружена на наш взгляд сенсационная связь взаимодействия спайк-белка капсида вируса COVID-19 на процесс одновременного образования тромбов и амилоида в цельной крови здоровых людей и перенесших COVID-19 [9]. Донорами цельной крови были выбраны предварительно обследованные относительно здоровые добровольцы без вредных привычек и заболеваний вен -трое мужчин и восемь женщин, средний возраст 43,4±11,7 года. ...
... Общая распространённость острых цереброваскулярных заболеваний при COVID-19 составила 2,3% (95% ДИ 1,0-3,6), из которых большинство составляли ишемический инсульт -2,1% (95% ДИ 0,9-3,3), геморрагический инсульт -0,4% (95% ДИ 0,2-0,6) и тромбоз церебральных вен -0,3% (95% ДИ 0,1-0,6). Среди других неврологических проявлений; нарушения обоняния -35,8% (95% ДИ 21,4-50,2), нарушения вкуса -38,5% (95% ДИ 24,0-53,0), миалгия -19,3% (95% ДИ 15,1-23,6), головная боль -14,7% (95% ДИ 10, [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]9), головокружение -6,1% (95% ДИ 3,1-9,2), -особо следует отметить синкопальные состояния, наблюдавшиеся у 1,8% (95% ДИ 0,9-4,6) пациентов, которые не описаны другими авторами и могут быть причиной внезапной смерти больных [64]. ...
We have considered the mechanisms of activation of coagulation hemostasis in COVID-19. It follows from the literature data that after interacting with the cell membrane of target cells, the spike proteins of the COVID-19 virus that have passed into the amyloid form initiate the formation of amyloid nanotubes from amyloid monomers and together activate plasma factor XI. This causes the formation of blood clots. The variety of neurological disorders in COVID-19 is provided by multiple thrombosis of small and large vessels of the central nervous system.
... Частицы S-белка способны взаимодействовать напрямую с тромбоцитами и белком фибриногеном, вызывая в нем изменения и, как следствие, нарушения свертываемости крови: формирование более плотных фибриновых сгустков, склеивание эритроцитов, гиперактивацию тромбоцитов и разрастание их мембраны. В присутствии тромбина спайковый белок способствует отложению фибрина амилоидной природы, формируя волокнистые и бесформенные сгустки и препятствуя току жидкости в сосудах [14]. ...
The ongoing COVID-19 pandemic has caused significant morbidity and mortality worldwide, as well as a profound impact on society. Among the nosologies that increase the risk of a severe course of COVID-19, coronary heart disease, chronic heart failure, cardiomyopathy. The main complications caused by coronavirus infection include thrombotic ones. Spike protein SARS-CoV-2 can interact directly with platelets and fibrin, causing blood hypercoagulation and obstructing blood flow. The presence of the spike protein in circulation leads to structural changes in fibrin, complement 3 and prothrombin, which can contribute to hypercoagulability in COVID-19 positive patients and cause a significant violation of fibrinolysis. Endothelial damage and systemic inflammation, being interrelated triggers of coagulopathy characteristic of COVID-19, trigger a cascade of reactions resulting in thrombotic complications against the background of endothelial dysfunction and hyperinflammation, which may be of clinical importance in the treatment of hypercoagulability in patients with COVID-19 (bibliography: 14 refs).
... Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 , is characterized by acute clinical pathologies, including various coagulopathies that may result in either bleeding and thrombocytopenia, hypercoagulation, pulmonary intravascular coagulation, microangiopathy venous thromboembolism or arterial thrombosis [1][2][3][4][5][6][7][8][9]. Acute COVID-19 infection is also characterized by dysregulated, circulating inflammatory biomarkers, hyperactivated platelets, damaged erythrocytes and substantial deposition of microclots in the lungs [6,[8][9][10][11][12][13][14][15][16]. ...
... The excitation wavelength for ThT was set at 450 nm to 488 nm and the emission at 499 nm to 529 nm and processed samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany) [5,8,9]. ...
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis.
Methods
We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms.
Results
We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits.
Conclusions
Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.
... In addition to trimerization, membrane anchoring seems to further improve immunogenicity, as transmembrane anchored prefusion-stabilized full-length S protein was reported to elicit higher VNA levels than corresponding secreted constructs [1,83]. Both in terms of immunogenicity and potential association of circulating SARS-Cov-2 S1 subunit with enhanced blood clotting [84], use of a small membrane-anchored antigen is rational. ...
Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.
A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal vs. pathologic convalescence process have not been well-delineated. We characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 4-6 weeks after initial clinical symptoms resolved. Convalescent subjects showed robust IgA and IgG responses and positive antibody correlations between matched saliva and plasma samples. However, global shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells and clotting factors in plasma (e.g., fibrinogen and antithrombin), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered pathways that correlated positively with IgA levels. Our study positions saliva as a viable fluid to monitor immunity beyond plasma to document COVID-19 immune, inflammatory, and coagulation-related sequelae.
State of the problem. Thrombotic complications are a common risk factor for a variety of diseases and are one of the leading causes of death. This leads to a strong interest in finding effective means of prevention and treatment. A characteristic feature of the last decade is the growing interest and numerous attempts to introduce into clinical practice fibrinolytic enzymes that are not functionally related to the hemostasis system. The aim of the work. Investigation of molecular mechanisms that cause the lack of efficiency of native fibrinolysis in relation to fibrin clots with impaired regularity of fibrin structure. Correlation of own achievements in the field of biochemistry and medicine with systematized literary material. The possibilities created by non-plasmin fibrinolytics and the advantages of their use are considered. Discussion and conclusions. The reasons of the complications caused by insufficient efficiency of both own fibrinolytic system, and the entered fibrinolytics are substantiated. It is shown that the leading role in such complications is played by violation of the regularity of the structure of fibrin clots. The mechanisms of action of leading fibrinolytic agents are considered and the expediency of using alternative non-plasmin fibrinolytics is substantiated. The properties and expediency of the use of fibrinolytics based on components of snake venoms and bacterial proteinases as effective means for the breakdown of fibrin with impaired regularity of structure are discussed. Key words: fibrosis, thrombotic therapy, fibrinolytic enzymes, hemostasis system.
Течение COVID-19 у больных нередко осложняется гиперкоагуляцией и тромбозами магистральных сосудов. Как оказалось, вакцинирование против COVID-19 препаратом ChAdOx1 nCoV-19 (AstraZeneca) у ряда пациентов также вызвало тромбоцитопению и тромбообразование в нетипичных (церебральный венозный синус, воротная, чревная, печеночная вены) и типичных (глубокие вены бедра и голени, тромбоэмболия легочной артерии, острые артериальные тромбозы) местах. А использование мРНК вакцины (Moderna и Pfizer) иногда сопровождалось тромбоцитопенией и кровотечением, но без образования тромбов. Это обстоятельство послужило по-водом для поиска механизмов тромбообразования при использовании ранее никогда не применявшихся вакцин, разработанных против COVID-19. Цель публикации-информирование врачебной общественности о механизмах тромбообразования при COVID-19; обсуждение возможных патогенетических путей быстрого образования амилоида и амилоидогенной стимуляции системы коагуляционного гемостаза. В единственном завершенном на настоящий момент исследовании представлены сведения о запуске спайк-белком капсида вируса COVID-19 быстрого формирования амилоида с образованием плотных крупных фибриновых сгустков в цельной крови как здоровых людей, так и у находившихся в остром периоде заболевания COVID-19. Авторы, обнаружив факт прямого влияния спайк-белка на формирование тромбов, тем не менее не исследовали возможные патогенетические пути запуска тромбо-образования спайк-белком. Поскольку авторы прямо указали на роль быстрого образования амилоида в запуске коагуляции, механизм которого неизвестен практикующим специалистам, имеет смысл обсудить вопросы быстрого образования амилоида в сосудистом русле и роли амилоида как фактора запуска коагуляционного гемостаза. Обсуждаемая публикация подтверждается ранее проведенными исследованиями других авторов о влиянии -амилоида и АА-амилоида на процессы образования тромбов при болезни Альцгеймера и системных амилоидозах. На основании изученных литературных источников нами высказано предположение, что у какой-то части больных, перенесших COVID-19 в тяжелой форме, в последующем может развиться системный амилоидоз. Введение. COVID-19 часто сопровождается клинически значимыми тромбозами, в том числе тромбоэмболией легочной артерии, частота которой в 3,4 раза выше у больных COVID-19, чем у пациентов с заболеваниями, тя-жесть которых аналогична [25]. По этой причине тема активно обсуждается в ряде научных статей [4-6]. Из данных этих статей следует, что основной при-чиной тромботических осложнений при COVID-19 являются диссеминирован-ное внутрисосудистое свертывание, тромботическая микроангиопатия и гепа-рин-индуцированная тромбоцитопения. В большинстве случаев наблюдается удлинение активированного частичного тромбопластинового времени, что яв-ляется свидетельством снижения активности фактора Хагемана (FXII).
