Relevance of coagulation parameters in long-COVID patients

Welcome back to this next session of the Hemostasis Learning Institute. My name is Doctor John Mitzios, Medical Sciences Partner at Siemens Health and Ears, and I'm the host of today's session. Today's speaker is Professor Razia Pretorius. Professor Pretorius is a professor and chair of the Physiological Science of Stellenbosch University in South Africa. She also has been named an an Honorary professor at Liverpool University in the UK. Her research spans hematology and systemic inflammation, aiming to reduce disease burden by identifying and targeting novel disease markers to mitigate and rest their effects on inflammation. She has authored or co-authored more than 300 peer reviewed papers. Today's presentation refers to the beginning of 2020 when SARS COV 2 appeared and changed our world. During that pandemic, billions were infected with SARS COV 2 and millions died from it. Soon, it became apparent that some people suffered from a wide range of ongoing health problems lasting weeks, months or even years. With millions of patients worldwide, long COVID or post COVID-19 syndrome remains a heavy burden from the pandemic. A better understanding of this phenomenon is urgently needed to develop therapies to help those affected. This lecture gives insight into the pathophysiology of long COVID and suggests treatment strategies for overcoming it. Professor Pretorius, the floor is yours. Hello everyone. Today, I will be sharing with you our latest research on clotting pathologies and I will spotlight our recent findings in COVID-19 as well as long COVID. I just want to show my disclosures. I was invited by Siemens Health and Years to attend this meeting and I have got various grants by Polybi Research Foundation, Bellevue Research Foundation, Kernels Funding Initiative, the NRA from South Africa and also the Medical Research Council from South Africa. The learning objectives today will be the relevance of receptor inflammatory marker interactions in driving disease pathologies, also the relevance of direct protein, protein interactions in clotting pathologies. And then I will end off by discussing a place of novel methods in the diagnosing of well known and new diseases. This is just my research collaborators and I wish to acknowledge them. It's Professor Douglas B Kell. He's from the University of Liverpool and he's a systems biologist, Various clinicians under the lead of Doctor Yaku Lobscher, the hematologist Dr. Jan Steenkamp, and various researchers as well as students. And then I also would like to say that Medi Clinic private hospital group approved sample collection at the facilities and we received ethics approval for the studies that will be discussed here. My lab and our collaborators from various laboratories over the world have been identifying in flammatory molecules in circulation that might be involved in and drive of pathological clotting. And we have also focused our research endeavours on studying the effects of increased circulating inflammatory molecules and how they interact with cells of the hematological system. We focus in particular on platelets and red blood cells as well as on the clotting protein fibrinogen. We are also interested in identifying novel inflammatory molecules that might play a role in persistent symptoms of long COVID. Over the years, we have published numerous papers that show the importance of inflammatory molecules in circulation and the role in abnormal blood clotting. I'm also interested in platelet signalling and the role in abnormal clotting. Platelets in circulation play a critical role in healthy blood clotting. However, they can become overstimulated and can drive pathological blood clotting if there are inflammatory molecules in circulation. This can also happen in the presence of viral infections, where platelets can act as important signalling entities. There is also complex relationship between receptors on platelets and endothelial cells we're circulating biomarkers may also bind to. As we now know, damaged endothelial cells and platelet hyperactivation are central in pathologies. Our research group have shown these pathologies in acute COVID-19, long COVID and in various published papers in the context of COVID-19. Platelets are therefore central in immune activation and general coagulation pathology and they can form various complexes and obviously also within cells. This is known as platelet clumping. We are also interested in pathological blood clotting involving the M clotting protein fibrinogen, which is a soluble protein in circulation when you are healthy. If you focus your attention to the three cartoons shown here on the left, Cartoon A is a healthy protein structure, which has many alpha quills and few beta sheets. However, in the presence of inflammation, oxidative stress and various circulating inflammatory molecules, this structure changes, where the inflammatory molecules also spike protein may bind to the fibrinogen protein shown in cartoon B. Such direct protein protein interactions may cause the alpha coils of the healthy clot or fibrillogen molecule to untwist into large beta sheets as seen in C in the cartoon on the far right. We suggest that fibrillogen molecules then have a fibrilloide structure and we have shown this with various amyloid fluorescent protein markers and one example of that that such a marker is few flavoured tea and also amitrackers. We call these fibrin alloy plasma protein microclots. We have lifted blood clotting in acute COVID. Using the scanning electron microscope, we found that platelets are damaged and clumped together and attached to red blood cells. The clotting proteins that are supposed to be soluble as just mentioned form spontaneous microclots. In the micrographs on the far left you can see platelets that are hyperactivated. Note the yellow arrows. In the middle are micrographs where platelets are attached to red blood cells. Note the white arrows. On the far right you will see micrographs of spontaneously formed microclots that even glue together red blood cells. Note the blue arrows. Now with the ultra structural pathology noted in scanning Electro microscopy in mind, we looked at platelet poor plasma and exposed it to our fluorescent marker CU flavin T which binds to damaged protein and also to open hydrophobic areas in protein that are damaged. This marker was first used to identify amyloid protein in brains of Alzheimer's disease patients many years ago. However, in 2016, our research group discovered that Theo flavin T also binds to misfolded fibrinogen and fibrin proteins during pathological blood clotting. Here you can see the difference in platelet poor plasma structure in acute COVID-19 patients, where we compared plasma smears of acute COVID-19 patients to that of healthy as well as diabetic patient plasma samples. Here we can see micro clot formation demarcated as the green signal from CU flavin T, our marker of misfolded protein. We found small areas of nearly abnormal clotlets, really small little signal that are present in plasma of healthy individuals. As can be seen in the micrographs on the left. More abnormal micro clots are typically noted in plasma of individuals with diabetes. What was, however, quite significant to us was the extent of the abnormal micro clots in acute COVID-19 plasma seen in the fluoride plate. Also note the scale bar of 10 micrometers at the bottom of the micro Groves. As microscopy results are difficult to quantify, we suggested a micro clot and platelet grading system based on various stages of severity of either micro clot activation or based on various stages of platelet activation. Here is an example of our platelet grading system. In our experiments, hematocrit samples are exposed to two fluorescent markers, CD62 PE, which is PE conjugated in the pink pinkish signal that is a marker of P selectin that is mentioned previously can be used if we see platelets are damaged. We also used pack one, which is the green signal that identifies platelets through marking the glycoprotein 2 beta 3A on the platelet membrane. The micrographs that you see here and the plate on the left and the top row shows examples of platelets from healthy or controlled samples with minimally activated platelets. This is stage one. This plate shows increased platelets spreading and the beginning of clumping. The micrograph plate on the right shows platelet clumping. Obviously in healthy individuals there will be no platelet clumping and therefore be marketed as none with A1. Use the platelet grading system. Here is our micro clot grading system shown on the left. Stage 1 and Stage 2 demarcates minimal micro clot formation in typically a healthy control sample. We also suggest that the various stages can be used in a numerical scoring system together with a platelet scoring system that might be used in both acute COVID-19 and long COVID. If you just note on this slide as well, just for comparison, I show microgloss again of the type 2 diabetes samples. So you could note the difference between diabetes and COVID. Now with our knowledge of acute COVID-19, we also turned our attention to the spike protein and also to the to the South, one of the spike protein in particular and also to long COVID. So what you can see here was an example of of some of our results that we published in 2021 where we studied the effect of the addition of the S1 of the spike protein on clotting when it is added to healthy blood samples. You can see here that both platelets are hyperactivated when spike protein is added and micro clots are also formed. If you look at these micrographs, you could see scanning electron microscopy results. On the left we we looked at the addition of our spike protein to to the to whole blood. At the top you could see healthy red blood cells as well as platelets. And when we added there is one protein part of the spike protein, we could induce significant microclotformation and platelet clumping and and also you could see how these platelets and microclods attached to the red blood cells. We also initiated A microfluidics experiment, see the plate on the right healthy plasma obviously as we know do not have much microclods and we also could not see this in a fluidic system. However, in both acute COVID-19 and this is the earlier Delta variant, significant clots are seen flowing the fluidic system. Such clutch in the micro fluidic system was also confirmed by our collaborator Martin Crater and he's at the Max Planck Institute. In fact, I'm very happy to report that he actually sent to US one of his micro fluidics instruments and that is now currently in our laboratory and we have been using it since January 2023. We could then, as you could see in this system at the bottom and if you can also see that we could indeed induce microclots that look very much, much similar to the acute COVID clots when we added spike protein to our healthy sample Recently together with collaborators from the USA, we also looked at micro clots and platelets from the more virulent beta and delta variants versus Omicron. And here you could see examples of our results. As noted in the top row. You can see that Omicron seems not to make so many micro plots as we have previously seen with the more virulent variants. However, platelet hyperactivation is not much different between the different variants where Omicron also causes significant platelet hyperactivation. Note, this is during the acute phase. Now we know there's a global catastrophe happening currently and that is long COVID. We lifted blood samples from patients and soon realized that they are persistent microclots and widespread endothelial dysfunction in long COVID patients. These pathologies we believe may be central in causing the widespread symptoms in these patients and may lead to tissue hypoxia. Here are examples of some of our analysis of our long COVID patients blood samples. You can once again here see various platelet hyper activations in the samples and you you can also see significant micro clot formation. We also have got a South African long COVID registry that was initiated in 2021 and you are just some of our samples that we published in 2021 on this long COVID registry. You can see here comorbidities as well as symptoms in our South African non COVID cohort and the the data that we published in that paper showed that it is not much different than what is seen in the rest of the world with the the most important comorbidity being high blood pressure, high cholesterol and symptoms being brain fog and concentration issues. Those are the main symptoms that patients struggle with. We also plan an experiment where we looked at proteomics and the reason for that was we wanted to see which molecules, which proteins are present in the plasma. And to our surprise, we found that the many of these molecules are actually not present as free molecules in the plasma, but draw that in trapped inside the microclods. So we plan the experiment where we used healthy plasma and type 2 diabetes samples that was stored and was before COVID-19 times. So they were stored in our freezer at in our -80 and we therefore know that those samples were not exposed to spike protein. And we compared it to acute COVID-19 and long COVID plasma samples. To our surprise, when we try to digest our sample, we found a visible deposit at the bottom of the little tube in the acute and the long coat samples, but not in the diabetes samples and also not in the control samples. If there was suggests that the proteins found inside the plasma of acute COVID and long COVID do not fully digest. However, controls and diabetes digest fully and degraded easily with a trypsin digestion process for proteomics. The sample supernatant was filtered and we viewed it after our first drive to with the first solubilisation step and we could indeed still see lots of micro clots not digested in the acute and the long COVID cohort. We then went a step further and eventually we could develop a second digestion step where we indeed could digest all our micro clots. And here is just some of the results that that we found. We found inside these micro clots. They were a substantially increased number of inflammatory molecules and obviously you would expect to see molecules like fibrinogen, the various chains. We found that we found serum amyloid A, we found von Willebrand factor, but one of the important findings was a substantial increase in A2 antiplasmin and that was quite significant for us because A2 antiplasmin plays an important role in the clotting cascade. If you look at this diagram here, I'm not going to go into detail of the clotting cascade, but the important place where A2 antiplasmin plays a role in the clotting cascade is that it prevents clot breakdown. And this gave us there for a reason to believe that the clots inside long COVID patients and probably also acute COVID do have a mechanism to to assist it not to breakdown to be in circulation for a much longer time. We also did a second proteomics analysis using a much larger cohort to sample. And in this analysis we found exactly the same type of molecules, but we also found some more interesting molecules. And interestingly we found a lot of molecules that have that point to that auto antibodies as well as antibodies are entrapped inside the clots. We also took the same samples and we analysed the soluble fraction. So the proteomics analysis was of the insoluble fraction and we also noted the main culprits which include A2 antiplasmin significantly increased. Now the question that arose is this is a microscopy method that we used initially for a grading system and the proteomics is, is a labour intensive, time intensive method to study micro clots and platelet hyperactivation using microscopy and that is probably not a feasible option. So we followed this up and we asked ourselves the question, is there a place for flow cytometry? And indeed there is. We have developed a method to detect micro clots inside a cell free plasma sample. So it's plated poor plasma and we could indeed, just as we saw under the fluorescent microscope using a imaging flow cytometer, develop a method to to detect clot load in blood samples or plasma samples where the cells were removed. I just want to show some of our recent results from a paper that we have published where we looked at. A clinician initiated treatment regime and this is patient data that shows patients with long COVID that had micro clots as well as platelet hyperactivation as seen with our microscopy method. If our our clinician treated these patients with a regime of triple therapy, which is anticoagulants to calm down platelets as well as to prevent micro clots to form that 80% of this cold reported self reported that they were feeling much better and the treatment response was quite good for us. Now all of these data together with this clinician initiated treatment regime point us to the fact that in long COVID we believe there's a a extreme clotting pathology and probably also ischemia reperfusion injury. And that is driven by both platelet hyperactivation, fibre and micro clots and all sorts of inflammatory molecules and antibodies. And that eventually we can suggest that if we look at long COVID, it might be related to a thrombotic endothelitis. Now we are very happy to say that in 2022, the US government document, the scale document shown here noted that micro clot presence, viral persistence, auto antibodies together with widespread organ damage should be recognized as central pathologist to look into in finding answers to long COVID. We also featured in science in in 2022 and also in August 2022 in Nature and recently in January 2023, our research was focused in National Geographic. Now coming back to the learning objectives of this presentation, I believe I showed good enough evidence that there is relevant receptive inflammatory marker interactions that drive long COVID. There's also a good amount of research pointing to the fact that there are direct protein, protein interactions and clotting pathologies in long COVID. And then the question to ask is, is there a novel? Is there a reason or a place for novel methodologies in diagnosing some diseases, new diseases and old diseases? And the the question to answer there is definitely there's a place for novel methodologies that might include imaging, flow cytometry, proteomics and fluorescent microscopy. However, those entities or those type of methods might be very tedious for a researcher to embark on. However, imaging flow cytometry might be used very successfully as a point of care type of of analysis in a pathology lab. But now the the question that further arises, is there a place for novel technologies like a chip or a, a lab on a chip type of analysis for microclods as well as platelet hyperactivation? And with that, I'll leave you and thank you very much for allowing me to present this to the Simmons housing. Yes.

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Invitation by Siemens Healthineers to attend this meeting · Polybio Research Foundation · Balvi Research Foundation · Kernls funding initiative Learning objectives Clotting in health and disease · Relevance of receptor-inflammatory marker interactions in driving disease pathologies · Relevance of direct protein-protein interactions in clotting pathologies · The place of novel methods in diagnosing well-known and new diseases YUNIVESITHI Research Collaborators · Prof Douglas B Kell: University of Liverpool (UK): Systems Biologist · Clinicians: Dr Jaco Laubscher: Mediclinic Stellenbosch (MMed: Internal Medicine); Dr Arneaux Kruger (private practice clinician) Haematologist: Dr Janami Steenkamp: Pathcare Dr Mare Vlok: Stellenbosch University (SA): Biochemist and Proteomics lab Massimo Nunes: PhD student Dr Chantelle Venter: Stellenbosch University (SA): Physiologist Dr Sunday Oladejo, Liam R. Watson, Prof Kanshu Rajaratnam and Prof Bruce W. Watson: Centre for Al Research, School for Data-Science & Computational Thinking, Stellenbosch · Mediclinic (private hospital group): approved sample collection at their facility Research Focus · Inflammatory molecules that may cause pathological clotting, including both viral and bacterial inflammagens Effects of circulating inflammatory molecules on platelets, RBCs and fibrin(ogen) the main clotting protein Trends in Endocrinology & Metabolism Platelets as Potent Signaling Entities in Type 2 Diabetes Mellitus Parkinson's Disease: A Systemic Inflammatory Disease Accompanied by Bacterial Inflammagens Seminars in Thrombosis and Haemostasis Pathological Clotting and Deep Vein Thrombosis THE LANCET in Patients with HIV NATURE REVIEWS @@ The effect of iron overload on red blood cell morphology Platelets: emerging facilitators SCIENTIFIC REPORTS of cellular crosstalk in rheumatoid arthritis OPEN Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation ORIGINAL INVESTIGATION Open Access INTERFACE Inflammatory cytokines in type 2 diabetes mellitus as facilitators of hypercoagulation and abnormal clot formation Platelets, circulating inflammatory molecules and abnormal blood clotting Platelet receptors Platelet/endothelial cell signalling Leukocytes Platelets Leukocytes cells CAM Platelets Science Platelets Interactions Platelet-Immune Cell Complexes Platelet clearance NETS Macrophage Platelet-activated GP neutrophil complex Activated Monocyte Platelet-activated T cell complex Platelet-monocyte complex CD40 Pathological Clotting Fibrinogen Healthy fibrin polymerization: Inflammatory molecules may Structural changes resulting in fibrinaloid many a-coils bind to the fibrinogen protein formation where a-coils untwist into ß- protein-protein interactions sheets Beta sheet Alpha Helix Pathological fibrin Random coils polymerization Kell DB, Pretorius E. Proteins behaving badly. Substoichiometric molecular control and amplification of the initiation and nature of amyloid fibril formation: lessons from and for blood clotting. Progress in Biophysics and Molecular Biology 2017; 123: 16-41. Platelet and clotting pathologies in acute COVID-19 Structural changes in platelets Microclots Low magnification Higher magnification 200 nm Structural Changes in Fibrin(ogen) Healthy Plasma Type 2 Diabetes Plasma COVID-19 Plasma A Microclot Grading System Spreading Clumping Non None CD62P (PE- conjugated) (Pinkish signal) - P-selectin PAC-1 (green signal) = glycoprotein platelet membrane. Laubscher GJ, Lourens PJ, Venter C, Kell DB, Pretorius E. TEG®, Microclot and Platelet Mapping for Guiding Early Management of Severe COVID-19 Coagulopathy. Journal of Clinical Medicine. 2021;10(22)doi:10.3390/jcm10225381 10um Laubscher GJ, Lourens PJ, Venter C, Kell DB, Pretorius E. TEG®, Microclot and Platelet Mapping for Guiding Early Management of Severe COVID-19 Coagulopathy. Journal of Clinical Medicine. 2021;10(22)doi:10.3390/jcm10225381 Spike protein S1 can induce fibrinolytic-resistant (Stellenbosch microclots and platelet hyperactivation Healthy PPP Healthy PPP + spike protein Healthy PPP + thrombin Healthy PPP + spike protein + thrombin 10 um Grobbelaar, L.M ., Venter, C ., Vlok, M ., Ngoepe, M ., Laubscher, G.J ., Lourens, P.J ., Steenkamp, J ., Kell, D.B ., and Pretorius, E. (2021). SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19. Biosci Rep 41. Scanning electron Microscopy and microfluidics: Spike protein S1 UNIVERSITY Healthy WB Spike Protein Spike Protein COVID-19 LIH Healthy plasma versus older beta/delta vs Omicron Microclots present in platelet poor plasma Control Grobbelaar, L.M ., Kruger, A ., Venter, C ., Burger, E.M ., Laubscher, G.J ., Maponga, T.G ., Kotze, M.J ., Kwaan, Platelets present in haematocrit H.C ., Miller, J.B ., Fulkerson, D ., et al. (2022). Relative BA 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. Semin Thromb Hemost 48, 858- A global catastrophe Long COVID cases: 100 000 000 Between 10%- 30% of Covid patients continue to have persistent, debilitating symptoms. No current diagnostic available for Long Covid/PASC Microclots and Platelets in Long COVID Long COVID Kruger, A ., Vlok, M ., Turner, S ., Venter, C ., Laubscher, G.J ., Kell, D.B ., and Pretorius, E. (2022). 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. Cardiovasc Diabetol 21, 190. SA Long COVID registry data Symptoms Co-morbidities Brain fog, concentration, forgetfulness High Blood Pressure Shortness of breath Constant fatigue Joint and muscle pain High Cholesterol Heart rate dysfunction Rheumatoid Arthritis Female Digestive problems Previous Blood Clots Loss of sense of smell and taste Diabetes-2 Depression and anxiety Cardiovascular disease Low Oxygen Levels Pretorius, E ., Venter, C ., Laubscher, G.J ., Auto-immune disease Recurring Chest Pains Kotze, M.J ., Oladejo, S.O ., Watson, L.R ., Rajaratnam, K ., Watson, B.W ., and Kell, Rosacea D.B. (2022). Prevalence of symptoms, Sleep disturbances Previous heart attack Male comorbidities, fibrin amyloid microclots and platelet pathology in individuals Psoriasis with Long COVID/Post-Acute Sequelae Previous Stroke Lupus of COVID-19 (PASC). Cardiovascular Kidney problems Gingivitis & Periodontitis Diabetology 21, 148. Proteomics of Plasma from Healthy, Diabetic, Acute COVID-19 and Long COVID Healthy Plasma and Type 2 plasma: degraded Proteomics filtered with Acute COVID-19 and C18 filter LongCOVID plasma Citrated Centrifuged: platelet poor Trypsin unfiltered blood degradation Visisible deposits Fluorescence microscopy 2021 Proteomics Analysis Digested pellet deposits (microclots) from acute COVID-19 samples vs digested plasma from Control samples These proteins are present in both sample types; and a fold change value more than 1 = the protein that more prevalent inside the digested pellet deposits from Long COVID/PASC samples. These proteins were concentrated inside the digested pellet deposits. Protein name Fold change P-value von Willebrand Factor Complement component C4b C-reactive protein Digested pellet deposits from Long COVID/PASC microclots samples vs digested plasma from Control samples Coagulation factor XIII A chain Plasminogen Fibrinogen alpha chain a2 antiplasmin Serum Amyloid A (SAA4) Digested pellet deposits from Long COVID/PASC microclots samples vs digested pellet deposits (microclots) from acute COVID-19 samples Fibrinogen a chain Coagulation factor XIII B Complement factor 1 Long COVID and trapped inflammatory molecules a2-antiplasmin (a2AP) inhibit plasmin and ultimately will prevent sufficient fibrinolysis from happening Intrinsic Pathway Extrinsic pathway molecules in Activated Protein C (APC) +Protein C regulation Thrombomodulin Common Pathway Crosslinked fibrin clot Fibrinolytic system Up-regulation Tissue plasminogen activator (TPA) and Urikinase-type plasminogen activator (uPA) Fibrin degradation products e.g. D-Dime Acute COVID-19 and Long COVID IXa Plasmin Xa Up n activator (TPA) and Tissue plasminogen a 2022 Proteomics Analysis Tissue damage Intrinsic pathway Contact and damaged vessels Factor VIII Kallikrein Factor XI Serpin Family C member 1 (AT3) Kinogen 1 B1/B2 (Bradykinin Tissue Factor Pathway Inhibitor (TFPI) Factor X + Factor IX Von Willebrand Factor Protein S Factor V UPA (Urikinase plasminogen activator) (Serpin family E member EPCR (Endothelial protein C receptor) PAR1 Platelet Platelet factor PCI (Serpin family A member 5) A1AT (Serpin family A member 1) Decreased PAR3, 4 activation a2AP (Serpin family F member 2) Carboxypeptidate B2(CPB2) A2M (a-2-macroglobulin) increaaed cross-linking increased VWF may lead to Fibrinaloid Fibrin Fibrinolyses failure clots monomers products Kruger, A ., Vlok, M ., Turner, S ., Venter, C ., Laubscher, G.J ., Kell, D.B ., and Pretorius, E. (2022). 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. Cardiovasc Diabetol 21, 190. fibrinolyses Decreased fibrinolyses SAA concentrations in Controls and Long COVID Controls and Long COVID Inflammatory Reference Controls (n=15): Long COVID(n=25): Unit Long Covid Control Long Covid Group molecule range Mean (SD) OR VWF antigen in Controls and Long COVID patients Median (Q1-Q3) SAA ** p<0.01 PF4 *p<0.05 VWF 55.9 - 161.6 E-selectin concentrations in Controls and Long Covid F PECAM-1 concentrations in Controls E-selectin ng.ml 1 PECAM-1 ng.ml-1 mg.L-1 Controls A place for flow cytometry? Unmasked Masked Microclots within area range Ch01 ns Count Area range(um2) 20um Clinician-initiated treatment regimens: patient data analysis Treatment Response Digestive Problems Long COVID symptoms Chest Pain Depression and/or Anxiety Sleep Disturbance Status Muscle and/or Improved Joint pain Palpitations No effect Svmotom Shortness of Breath after treatment Cognitive Dysfunction Fatigue ment No improve- % Improvement of symptoms Co-morbidities before acute COVID-19 infection Number of Patients Ischaemia-reperfusion (I-R) injury Spike protein Endothelial pathology Inflammation/cytokine hyperactivation production Fibrin amyloid Oxidative stress microclots Ischaemia-reperfusion Genetic predisposition injury Lifestyle factors Kell, D.B ., and Pretorius, E. (2022). 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. Biochem J 479, 1653-1708. ation/ oduct tivatio mic ative -re festy Thrombotic Endothelialitis US GAO February 2022 GAO Science, Technology Assessment, and Analytics MARCH 2022 WHY THIS MATTERS Long COVID has potentially affected up to 23 million SCIENCE & TECH SPOTLIGHT: Americans, pushing an estimated 1 million people out of work. The full magnitude of health and economic effects LONG COVID is unknown but is expected to be significant. The causes of long COVID are not fully understood, complicating diagnosis and treatment. The condition raises policy questions, such as how best to support patients. Autoimmune Persistent Organ response virus damage Source: GAO analysis of medical literature. | GAO-22-105666 Science feature: June 2022 FEATURES CLUES TO LONG COVID Scientists strive to unravel what is driving disabling symptoms NEWS FEATURE: NATURE 24 August 2022 Could tiny blood clots cause long COVID's puzzling symptoms? NATIONAL GEOGRAPHIC JANUARY 2023 NATIONAL GEOGRAPHIC AND RESIA PRETORIUS 'Microclots' could help solve the long COVID puzzle Disease-associated inflammatory molecules in circulation Receptor-mediated response Protein-protein mediated response · Relevance of receptor- inflammatory marker interactions in driving disease pathologies · Relevance of direct protein- protein interactions in clotting · The place of novel methods in A place of novel methodologies diagnosing well-known and new diseases Fluorescence microscopy: automation Flow cytometry A place for novel technologies? Thank you Enkosi Dankie Photo by Justin Alberts Hea Siemens Healthcare Diagnostics Inc ., 2025 The products and features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed. SLS-24-3801-76 QR700016261