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.

Background Fibrin(ogen) amyloid microclots and platelet hyperactivation previously reported as a novel finding in South African patients with the coronavirus 2019 disease (COVID-19) and Long COVID/Post-Acute Sequelae of COVID-19 (PASC), might form a suitable set of foci for the clinical treatment of the symptoms of Long COVID/PASC. A Long COVID/PASC Registry was subsequently established as an online platform where patients can report Long COVID/PASC symptoms and previous comorbidities. Methods In this study, we report on the comorbidities and persistent symptoms, using data obtained from 845 South African Long COVID/PASC patients. By using a previously published scoring system for fibrin amyloid microclots and platelet pathology, we also analysed blood samples from 80 patients, and report the presence of significant fibrin amyloid microclots and platelet pathology in all cases. Results Hypertension, high cholesterol levels (dyslipidaemia), cardiovascular disease and type 2 diabetes mellitus (T2DM) were found to be the most important comorbidities. The gender balance (70% female) and the most commonly reported Long COVID/PASC symptoms (fatigue, brain fog, loss of concentration and forgetfulness, shortness of breath, as well as joint and muscle pains) were comparable to those reported elsewhere. These findings confirmed that our sample was not atypical. Microclot and platelet pathologies were associated with Long COVID/PASC symptoms that persisted after the recovery from acute COVID-19. Conclusions Fibrin amyloid microclots that block capillaries and inhibit the transport of O2 to tissues, accompanied by platelet hyperactivation, provide a ready explanation for the symptoms of Long COVID/PASC. Removal and reversal of these underlying endotheliopathies provide an important treatment option that urgently warrants controlled clinical studies to determine efficacy in patients with a diversity of comorbidities impacting on SARS-CoV-2 infection and COVID-19 severity. We suggest that our platelet and clotting grading system provides a simple and cost-effective diagnostic method for early detection of Long COVID/PASC as a major determinant of effective treatment, including those focusing on reducing clot burden and platelet hyperactivation.

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 S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation.  Using platelet poor plasma (PPP), we show that spike protein may interfere with blood flow.  Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.

Background Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. Methods Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. Results Our long COVID cohort’s symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. Conclusion Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Progressive respiratory failure is seen as a major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection. Relatively little is known about the associated morphologic and molecular changes in the circulation of these patients. In particular, platelet and erythrocyte pathology might result in severe vascular issues, and the manifestations may include thrombotic complications. These thrombotic pathologies may be both extrapulmonary and intrapulmonary and may be central to respiratory failure. Previously, we reported the presence of amyloid microclots in the circulation of patients with coronavirus disease 2019 (COVID-19). Here, we investigate the presence of related circulating biomarkers, including C-reactive protein (CRP), serum ferritin, and P-selectin. These biomarkers are well-known to interact with, and cause pathology to, platelets and erythrocytes. We also study the structure of platelets and erythrocytes using fluorescence microscopy (using the markers PAC-1 and CD62PE) and scanning electron microscopy. Thromboelastography and viscometry were also used to study coagulation parameters and plasma viscosity. We conclude that structural pathologies found in platelets and erythrocytes, together with spontaneously formed amyloid microclots, may be central to vascular changes observed during COVID-19 progression, including thrombotic microangiopathy, diffuse intravascular coagulation, and large-vessel thrombosis, as well as ground-glass opacities in the lungs. Consequently, this clinical snapshot of COVID-19 strongly suggests that it is also a true vascular disease and considering it as such should form an essential part of a clinical treatment regime.