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 Role of Fibrinolytic System in Health and Disease.

The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders.

Associations between hemostatic markers and mortality in COVID-19 - Compounding effects of D-dimer, antithrombin and PAP complex.

In this single-center cohort study, we applied a panel of laboratory markers to characterize hemostatic function in 217 consecutive patients that underwent testing for COVID-19 as they were admitted to Linköping University Hospital between April and June 2020. In the 96 patients that tested positive for SARS-CoV-2 (COVID-19+), the cumulative incidences of death and venous thromboembolism were 24.0% and 19.8% as compared to 12.4% (p = 0.031) and 11.6% (p = 0.13) in the 121 patients that tested negative (COVID-19-). In COVID-19+ patients, we found pronounced increases in plasma levels of von Willebrand factor (vWF) and fibrinogen. Excess mortality was observed in COVID-19+ patients with the following aberrations in hemostatic markers: high D-dimer, low antithrombin or low plasmin-antiplasmin complex (PAP) formation, with Odds Ratios (OR) for death of 4.7 (95% confidence interval (CI95) 1.7-12.9; p = 0.003) for D-dimer >0.5 mg/L, 5.9 (CI95 1.8-19.7; p = 0.004) for antithrombin (AT) ˂0.85 kIU/l and 4.9 (CI95 1.3-18.3; p = 0.019) for PAP < 1000 µg/L. Compounding increases in mortality was observed in COVID-19+ patients with combined defects in markers of fibrinolysis and coagulation, with ORs for death of 15.7 (CI95 4.3-57; p < 0.001) for patients with PAP <1000 µg/L and D-dimer >0.5 mg/L and 15.5 (CI95 2.8-87, p = 0.002) for patients with PAP <1000 µg/L and AT ˂0.85 kIU/L. We observed an elevated fraction of incompletely degraded D-dimer fragments in COVID-19+ patients with low PAP, indicating impaired fibrinolytic breakdown of cross-linked fibrin.

Therapeutic Strategies for Disseminated Intravascular Coagulation Associated with Aortic Aneurysm.

Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α2 plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.

Distinctive Biomarker Features in the Endotheliopathy of COVID-19 and Septic Syndromes.

BACKGROUND: Endotheliopathy is a key element in COVID-19 pathophysiology, contributing to both morbidity and mortality. Biomarkers distinguishing different COVID-19 phenotypes from sepsis syndrome remain poorly understood. OBJECTIVE: To characterize circulating biomarkers of endothelial damage in different COVID-19 clinical disease stages compared with sepsis syndrome and normal volunteers. METHODS: Patients with COVID-19 pneumonia (n = 49) were classified into moderate, severe, or critical (life-threatening) disease. Plasma samples were collected within 48 to 72 h of hospitalization to analyze endothelial activation markers, including soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), von Willebrand Factor (VWF), A disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 (ADAMTS-13) activity, thrombomodulin (TM), and soluble TNF receptor I (sTNFRI); heparan sulfate (HS) for endothelial glycocalyx degradation; C5b9 deposits on endothelial cells in culture and soluble C5b9 for complement activation; circulating dsDNA for neutrophil extracellular traps (NETs) presence, and α2-antiplasmin and PAI-1 as parameters of fibrinolysis. We compared the level of each biomarker in all three COVID-19 groups and healthy donors as controls (n = 45). Results in critically ill COVID-19 patients were compared with other intensive care unit (ICU) patients with septic shock (SS, n = 14), sepsis (S, n = 7), and noninfectious systemic inflammatory response syndrome (NI-SIRS, n = 7). RESULTS: All analyzed biomarkers were increased in COVID-19 patients versus controls (P < 0.001), except for ADAMTS-13 activity that was normal in both groups. The increased expression of sVCAM-1, VWF, sTNFRI, and HS was related to COVID-19 disease severity (P < 0.05). Several differences in these parameters were found between ICU groups: SS patients showed significantly higher levels of VWF, TM, sTNFRI, and NETS compared with critical COVID-19 patients and ADAMTS-13 activity was significantly lover in SS, S, and NI-SIRS versus critical COVID-19 (P < 0.001). Furthermore, α2-antiplasmin activity was higher in critical COVID-19 versus NI-SIRS (P < 0.01) and SS (P < 0.001), whereas PAI-1 levels were significantly lower in COVID-19 patients compared with NI-SIRS, S, and SS patients (P < 0.01). CONCLUSIONS: COVID-19 patients present with increased circulating endothelial stress products, complement activation, and fibrinolytic dysregulation, associated with disease severity. COVID-19 endotheliopathy differs from SS, in which endothelial damage is also a critical feature of pathobiology. These biomarkers could help to stratify the severity of COVID-19 disease and may also provide information to guide specific therapeutic strategies to mitigate endotheliopathy progression.

Characterization of fibrin/fibrinogen degradation products reagents and their utility in critical care patients with enhanced fibrinolysis.

INTRODUCTION: Fibrin/fibrinogen degradation products (FDP) values reflect coagulation and fibrinolysis status, and FDP levels are helpful for diagnosis and classification of disseminated intravascular coagulation (DIC). FDP measurement has always played a key role in diagnosing DIC, a phenomenon that has recently gained renewed attention because of its occurrence in coronavirus disease 2019 (COVID-19) patients. Although the evaluation of FDP is crucial for the management of critical care, the variability among FDP reagents is unclear. In this study, we aimed to compare LIASAUTO P-FDP with three FDP reagents and investigate their characteristics. METHODS: In total, 172 plasmas samples were used in the correlation. The sample data were divided into three groups including negative, no and positive discrepancy based on the discrepancy percentages calculated from each correlation between LIASAUTO P-FDP and other three reagents. D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), fibrinogen (Fbg) and Plasmin-α2 Plasmin Inhibitor (α2 PI) were measured and included in data analysis. RESULTS: The positive discrepancy groups showed higher D-dimer, PIC and FMC values than the negative discrepancy groups. The data indicated that LIASAUTO P-FDP had higher reactivity to D-dimer than other reagents and the values were elevated in the fibrinolysis-enhanced samples with various FDP fragments. CONCLUSION: LIASAUTO P-FDP displayed the reactivity towards various fibrin/fibrinogen degradation products, and it might be useful for DIC diagnosis because the fibrinolytic status differed in the DIC types and stages.