Data sharing: A Long COVID perspective, challenges, and road map for the future

The article focuses on the need for standardized data collection, interpretation, and reporting in the management of Long COVID. Topics include the lack of a consistent definition for Long COVID, the benefits of adopting a data-driven framework for Long COVID management, and the challenges and strategies of data sharing in Long COVID research.

Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis.

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome.

INTRODUCTION: The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. OBJECTIVES: Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient's infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). METHODS: High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. RESULTS: The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74-0.91) and 0.76 (CI 0.67-0.86). CONCLUSION: Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

Long COVID: pathophysiological factors and abnormalities of coagulation.

Acute COVID-19 infection is followed by prolonged symptoms in approximately one in ten cases: known as Long COVID. The disease affects ~65 million individuals worldwide. Many pathophysiological processes appear to underlie Long COVID, including viral factors (persistence, reactivation, and bacteriophagic action of SARS CoV-2); host factors (chronic inflammation, metabolic and endocrine dysregulation, immune dysregulation, and autoimmunity); and downstream impacts (tissue damage from the initial infection, tissue hypoxia, host dysbiosis, and autonomic nervous system dysfunction). These mechanisms culminate in the long-term persistence of the disorder characterized by a thrombotic endothelialitis, endothelial inflammation, hyperactivated platelets, and fibrinaloid microclots. These abnormalities of blood vessels and coagulation affect every organ system and represent a unifying pathway for the various symptoms of Long COVID.

Cardiovascular and haematological pathology in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): A role for viruses.

ME/CFS is a debilitating chronic condition that often develops after viral or bacterial infection. Insight from the study of Long COVID/Post Acute Sequelae of COVID-19 (PASC), the post-viral syndrome associated with SARS-CoV-2 infection, might prove to be useful for understanding pathophysiological mechanisms of ME/CFS. Disease presentation is similar between the two conditions, and a subset of Long COVID patients meet the diagnostic criteria for ME/CFS. Since Long COVID is characterized by significant vascular pathology - including endothelial dysfunction, coagulopathy, and vascular dysregulation - the question of whether or not the same biological abnormalities are of significance in ME/CFS arises. Cardiac abnormalities have for a while now been documented in ME/CFS cohorts, with recent studies demonstrating major deficits in cerebral blood flow, and hence vascular dysregulation. A growing body of research is demonstrating that ME/CFS is accompanied by platelet hyperactivation, anomalous clotting, a procoagulant phenotype, and endothelial dysfunction. Endothelial damage and dysregulated clotting can impair substance exchange between blood and tissues, and result in hypoperfusion, which may contribute to the manifestation of certain ME/CFS symptoms. Here we review the ME/CFS literature to summarize cardiovascular and haematological findings documented in patients with the condition, and, in this context, briefly discuss the potential role of previously-implicated pathogens. Overall, cardiac and haematological abnormalities are present within ME/CFS cohorts. While atherosclerotic heart disease is not significantly associated with ME/CFS, suboptimal cardiovascular function defined by reduced cardiac output, impaired cerebral blood flow, and vascular dysregulation are, and these abnormalities do not appear to be influenced by deconditioning. Rather, these cardiac abnormalities may result from dysfunction in the (autonomic) nervous system. Plenty of recently published studies are demonstrating significant platelet hyperactivity and endothelial dysfunction in ME/CFS, as well as anomalous clotting processes. It is of particular importance to determine to what extent these cardiovascular and haematological abnormalities contribute to symptom severity, and if these two systems can be targeted for therapeutic purposes. Viral reservoirs of herpesviruses exist in ME/CFS, and most likely contribute to cardiovascular and haematological dysfunction directly or indirectly. This review highlights the potential of studying cardiac functioning, the vasculature, and coagulation system in ME/CFS.

Relative Hypercoagulopathy of the SARS-CoV-2 Beta and Delta Variants when Compared to the Less Severe Omicron Variants Is Related to TEG Parameters, the Extent of Fibrin Amyloid Microclots, and the Severity of Clinical Illness.

Earlier variants of SARS-CoV-2 have been associated with hypercoagulability and an extensive formation of fibrin amyloid microclots, which are considered to contribute to the pathology of the coronavirus 2019 disease (COVID-19). The newer omicron variants appear to be far more transmissible, but less virulent, even when taking immunity acquired from previous infections or vaccination into account. We here show that while the clotting parameters associated with omicron variants are significantly raised over those of healthy, matched controls, they are raised to levels significantly lower than those seen with more severe variants such as beta and delta. We also observed that individuals infected with omicron variants manifested less extensive microclot formation in platelet-poor plasma compared with those harboring the more virulent variants. The measurement of clotting effects between the different variants acts as a kind of "internal control" that demonstrates the relationship between the extent of coagulopathies and the virulence of the variant of interest. This adds to the evidence that microclots may play an important role in reflecting the severity of symptoms observed in COVID-19.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system.

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.

The potential role of ischaemia-reperfusion injury in chronic, relapsing diseases such as rheumatoid arthritis, Long COVID, and ME/CFS: evidence, mechanisms, and therapeutic implications.

Ischaemia-reperfusion (I-R) injury, initiated via bursts of reactive oxygen species produced during the reoxygenation phase following hypoxia, is well known in a variety of acute circumstances. We argue here that I-R injury also underpins elements of the pathology of a variety of chronic, inflammatory diseases, including rheumatoid arthritis, ME/CFS and, our chief focus and most proximally, Long COVID. Ischaemia may be initiated via fibrin amyloid microclot blockage of capillaries, for instance as exercise is started; reperfusion is a necessary corollary when it finishes. We rehearse the mechanistic evidence for these occurrences here, in terms of their manifestation as oxidative stress, hyperinflammation, mast cell activation, the production of marker metabolites and related activities. Such microclot-based phenomena can explain both the breathlessness/fatigue and the post-exertional malaise that may be observed in these conditions, as well as many other observables. The recognition of these processes implies, mechanistically, that therapeutic benefit is potentially to be had from antioxidants, from anti-inflammatories, from iron chelators, and via suitable, safe fibrinolytics, and/or anti-clotting agents. We review the considerable existing evidence that is consistent with this, and with the biochemical mechanisms involved.

Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-Acute Sequelae of COVID-19 (PASC).

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.

The Occurrence of Hyperactivated Platelets and Fibrinaloid Microclots in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

We have previously demonstrated that platelet-poor plasma (PPP) obtained from patients with Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is characterized by a hypercoagulable state and contains hyperactivated platelets and considerable numbers of already-formed amyloid fibrin(ogen) or fibrinaloid microclots. Due to the substantial overlap in symptoms and etiology between Long COVID/PASC and ME/CFS, we investigated whether coagulopathies reflected in Long COVID/PASC-hypercoagulability, platelet hyperactivation, and fibrinaloid microclot formation-were present in individuals with ME/CFS and gender- and age-matched healthy controls. ME/CFS samples showed significant hypercoagulability as judged by thromboelastography of both whole blood and platelet-poor plasma. The area of plasma images containing fibrinaloid microclots was commonly more than 10-fold greater in untreated PPP from individuals with ME/CFS than in that of healthy controls. A similar difference was found when the plasma samples were treated with thrombin. Using fluorescently labelled PAC-1, which recognizes glycoprotein IIb/IIIa, and CD62P, which binds P-selectin, we observed hyperactivation of platelets in ME/CFS hematocrit samples. Using a quantitative scoring system, the ME/CFS platelets were found to have a mean spreading score of 2.72 ± 1.24 vs. 1.00 (activation with pseudopodia formation) for healthy controls. We conclude that ME/CFS is accompanied by substantial and measurable changes in coagulability, platelet hyperactivation, and fibrinaloid microclot formation. However, the fibrinaloid microclot load was not as great as was previously noted in Long COVID/PASC. Fibrinaloid microclots, in particular, may contribute to many ME/CFS symptoms, such as fatigue, seen in patients with ME/CFS, via the (temporary) blockage of microcapillaries and hence ischemia. Furthermore, fibrinaloid microclots might damage the endothelium. The discovery of these biomarkers represents an important development in ME/CFS research. It also points to possible uses for treatment strategies using known drugs and/or nutraceuticals that target systemic vascular pathology and endothelial inflammation.

Immuno-Thrombotic Complications of COVID-19: Implications for Timing of Surgery and Anticoagulation.

Early in the coronavirus disease 2019 (COVID-19) pandemic, global governing bodies prioritized transmissibility-based precautions and hospital capacity as the foundation for delay of elective procedures. As elective surgical volumes increased, convalescent COVID-19 patients faced increased postoperative morbidity and mortality and clinicians had limited evidence for stratifying individual risk in this population. Clear evidence now demonstrates that those recovering from COVID-19 have increased postoperative morbidity and mortality. These data-in conjunction with the recent American Society of Anesthesiologists guidelines-offer the evidence necessary to expand the early pandemic guidelines and guide the surgeon's preoperative risk assessment. Here, we argue elective surgeries should still be delayed on a personalized basis to maximize postoperative outcomes. We outline a framework for stratifying the individual COVID-19 patient's fitness for surgery based on the symptoms and severity of acute or convalescent COVID-19 illness, coagulopathy assessment, and acuity of the surgical procedure. Although the most common manifestation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19 pneumonitis, every system in the body is potentially afflicted by an endotheliitis. This endothelial derangement most often manifests as a hypercoagulable state on admission with associated occult and symptomatic venous and arterial thromboembolisms. The delicate balance between hyper and hypocoagulable states is defined by the local immune-thrombotic crosstalk that results commonly in a hemostatic derangement known as fibrinolytic shutdown. In tandem, the hemostatic derangements that occur during acute COVID-19 infection affect not only the timing of surgical procedures, but also the incidence of postoperative hemostatic complications related to COVID-19-associated coagulopathy (CAC). Traditional methods of thromboprophylaxis and treatment of thromboses after surgery require a tailored approach guided by an understanding of the pathophysiologic underpinnings of the COVID-19 patient. Likewise, a prolonged period of risk for developing hemostatic complications following hospitalization due to COVID-19 has resulted in guidelines from differing societies that recommend varying periods of delay following SARS-CoV-2 infection. In conclusion, we propose the perioperative, personalized assessment of COVID-19 patients' CAC using viscoelastic hemostatic assays and fluorescent microclot analysis.

A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications.

Post-acute sequelae of COVID (PASC), usually referred to as 'Long COVID' (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, 'brain fog', tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection. It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of 'COVID', although its demographics are quite different from those of acute COVID-19. A few years ago, we discovered that fibrinogen in blood can clot into an anomalous 'amyloid' form of fibrin that (like other ß-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies. These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope. Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms. Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored 'triple' anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.

TEG®, Microclot and Platelet Mapping for Guiding Early Management of Severe COVID-19 Coagulopathy.

An important component of severe COVID-19 disease is virus-induced endothelilitis. This leads to disruption of normal endothelial function, initiating a state of failing normal clotting physiology. Massively increased levels of von Willebrand Factor (VWF) lead to overwhelming platelet activation, as well as activation of the enzymatic (intrinsic) clotting pathway. In addition, there is an impaired fibrinolysis, caused by, amongst others, increased levels of alpha-(2) antiplasmin. The end result is hypercoagulation (proven by thromboelastography® (TEG®)) and reduced fibrinolysis, inevitably leading to a difficult-to-overcome hypercoagulated physiological state. Platelets in circulation also plays a significant role in clot formation, but they themselves may also drive hypercoagulation when they are overactivated due to the interactions of their receptors with the endothelium, immune cells or circulating inflammatory molecules. From the literature it is clear that the role of platelets in severely ill COVID-19 patients has been markedly underestimated or even ignored. We here highlight the value of early management of severe COVID-19 coagulopathy as guided by TEG®, microclot and platelet mapping. We also argue that the failure of clinical trials, where the efficacy of prophylactic versus therapeutic clexane (low molecular weight heparin (LMWH)) were not always successful, which may be because the significant role of platelet activation was not taken into account during the planning of the trial. We conclude that, because of the overwhelming alteration of clotting, the outcome of any trial evaluating an any single anticoagulant, including thrombolytic, would be negative. Here we suggest the use of the degree of platelet dysfunction and presence of microclots in circulation, together with TEG®, might be used as a guideline for disease severity. A multi-pronged approach, guided by TEG® and platelet mapping, would be required to maintain normal clotting physiology in severe COVID-19 disease.

Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin.

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.

Erythrocyte, Platelet, Serum Ferritin, and P-Selectin Pathophysiology Implicated in Severe Hypercoagulation and Vascular Complications in COVID-19.

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.

SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19.

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 ß 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.

Prevalence of readily detected amyloid blood clots in 'unclotted' Type 2 Diabetes Mellitus and COVID-19 plasma: a preliminary report.

BACKGROUND: Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. METHODS: Platelet poor plasma (PPP) was collected and frozen. On the day of analysis, PPP samples were thawed and analysed. We show here that microclots can be detected in the native plasma of twenty COVID-19, as well as ten T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results were compared to ten healthy age-matched individuals. RESULTS: In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. CONCLUSIONS: This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P < 0.0001) and is consistent with the recognition that the early detection and prevention of such clotting can have an important role in therapy.

Covid-19: The Rollercoaster of Fibrin(Ogen), D-Dimer, Von Willebrand Factor, P-Selectin and Their Interactions with Endothelial Cells, Platelets and Erythrocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), also known as coronavirus disease 2019 (COVID-19)-induced infection, is strongly associated with various coagulopathies that may result in either bleeding and thrombocytopenia or hypercoagulation and thrombosis. Thrombotic and bleeding or thrombotic pathologies are significant accompaniments to acute respiratory syndrome and lung complications in COVID-19. Thrombotic events and bleeding often occur in subjects with weak constitutions, multiple risk factors and comorbidities. Of particular interest are the various circulating inflammatory coagulation biomarkers involved directly in clotting, with specific focus on fibrin(ogen), D-dimer, P-selectin and von Willebrand Factor (VWF). Central to the activity of these biomarkers are their receptors and signalling pathways on endothelial cells, platelets and erythrocytes. In this review, we discuss vascular implications of COVID-19 and relate this to circulating biomarker, endothelial, erythrocyte and platelet dysfunction. During the progression of the disease, these markers may either be within healthy levels, upregulated or eventually depleted. Most significant is that patients need to be treated early in the disease progression, when high levels of VWF, P-selectin and fibrinogen are present, with normal or slightly increased levels of D-dimer (however, D-dimer levels will rapidly increase as the disease progresses). Progression to VWF and fibrinogen depletion with high D-dimer levels and even higher P-selectin levels, followed by the cytokine storm, will be indicative of a poor prognosis. We conclude by looking at point-of-care devices and methodologies in COVID-19 management and suggest that a personalized medicine approach should be considered in the treatment of patients.

The Biology of Lactoferrin, an Iron-Binding Protein That Can Help Defend Against Viruses and Bacteria.

Lactoferrin is a nutrient classically found in mammalian milk. It binds iron and is transferred via a variety of receptors into and between cells, serum, bile, and cerebrospinal fluid. It has important immunological properties, and is both antibacterial and antiviral. In particular, there is evidence that it can bind to at least some of the receptors used by coronaviruses and thereby block their entry. Of importance are Heparan Sulfate Proteoglycans (HSPGs) and the host receptor angiotensin-converting enzyme 2 (ACE2), as based on other activities lactoferrin might prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from attaching to the host cells. Lactoferrin (and more specifically enteric-coated LF because of increased bioavailability) may consequently be of preventive and therapeutic value during the present COVID-19 pandemic.