Assays to quantify fibrinolysis: strengths and limitations. Communication from the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee on fibrinolysis.

Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease plasmin, which cleaves and solubilizes crosslinked fibrin clots into fibrin degradation products. The quantity and quality of fibrinolytic enzymes, their respective inhibitors, and clot structure determine overall fibrinolysis. The quantity of protein can be measured by antigen-based assays, and both quantity and quality can be assessed using functional assays. Furthermore, variations of commonly used assays have been reported, which are tailored to address the role(s) of specific fibrinolytic factors and cellular elements (eg, platelets, neutrophils, and red blood cells). Although the concentration and/or activity of a protein can be quantified, how these individual components contribute to the overall fibrinolysis outcome can be challenging to determine. This difficulty is due to temporal changes within and around the thrombi during the clot breakdown, particularly the fibrin matrix structure, and composition. Furthermore, terms such as "fibrinolytic activity/potential," "plasminogen activation," and "plasmin activity" are often used interchangeably despite having different definitions. The purpose of this review is to 1) summarize the assays measuring fibrinolysis activity and potential, 2) facilitate the interpretation of data generated by these assays, and 3) summarize the strengths and limitations of these assays.

TFPI and FXIII negatively and S100A8/A9 and Cystatin C positively correlate with D-dimer in COVID-19.

D-dimer is an established biomarker of thromboembolism and severity in COVID-19. We and others have recently reported the dysregulation of tissue factor pathway inhibitor (TFPI), FXIII, fibrinolytic pathway, inflammatory markers, and tissue injury markers, particularly in severe COVID-19. However, association of these markers with thromboembolism in COVID-19 remains elusive. The correlation analyses between these markers in patients with moderate (non-ICU) and severe COVID-19 (ICU) were performed to delineate the potential pathomechanisms and impact of thromboembolism. We observe a negative correlation of plasma TFPI (r2 = 0.148, P = 0.035), FXIII (r2 = 0.242, P = 0.006), and plasminogen (r2 = 0.27, P = 0.003) with D-dimer, a biomarker of thromboembolism, levels in these patients. Further analysis revealed a strong positive correlation between fibrinolytic markers tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) (r2 = 0.584, P < 0.0001). Interestingly, a significant positive correlation of PAI-1, but not tPA, was observed with platelets and endothelial cells dysfunction markers P-selectin (r2 = 0.184, P = 0.01) and soluble CD40 ligand (sCD40 L) (r2 = 0.163, P = 0.02). Moreover, calprotectin (S100A8/A9) and cystatin C (CST3), previously linked with thromboembolism, exhibited positive correlations with each other (r2 = 0.339, P = 0.0007) and with the level of D-dimer independently in COVID-19. Finally, the tissue injury marker myoglobin demonstrated a strong positive correlation with D-dimer (r2 = 0.408, P = 0.0001). Taken together, inverse correlations of TFPI and FXIII with D-dimer suggest the TF pathway activation and aberrant fibrin polymerization in COVID-19 patients. The elevated level of PAI-1 is potentially contributed by activated platelets and endothelial cells. S100A8/A9 may also play roles in impaired fibrinolysis and thromboembolism, in part, through regulating the CST3. These findings strengthen the understanding of thromboembolism and tissue injury and may help in better management of thromboembolic complications in COVID-19 patients.

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.

Altered fibrin network structure and fibrinolysis in intensive care unit patients with COVID-19, not entirely explaining the increased risk of thrombosis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 infection is associated with an increased incidence of thrombosis. OBJECTIVES: By studying the fibrin network structure of coronavirus disease 2019 (COVID-19) patients, we aimed to unravel pathophysiological mechanisms that contribute to this increased risk of thrombosis. This may contribute to optimal prevention and treatment of COVID-19 related thrombosis. PATIENTS/METHODS: In this case-control study, we collected plasma samples from intensive care unit (ICU) patients with COVID-19, with and without confirmed thrombosis, between April and December 2020. Additionally, we collected plasma from COVID-19 patients admitted to general wards without thrombosis, from ICU patients with pneumococcal infection, and from healthy controls. Fibrin fiber diameters and fibrin network density were quantified in plasma clots imaged with stimulated emission depletion microscopy and confocal microscopy. Finally, we determined the sensitivity to fibrinolysis. RESULTS: COVID-19 ICU patients (n = 37) and ICU patients with pneumococcal disease (n = 7) showed significantly higher fibrin densities and longer plasma clot lysis times than healthy controls (n = 7). No differences were observed between COVID-19 ICU patients with and without thrombosis, or ICU patients with pneumococcal infection. At a second time point, after diagnosis of thrombosis or at a similar time point in patients without thrombosis, we observed thicker fibers and longer lysis times in COVID-19 ICU patients with thrombosis (n = 19) than in COVID-19 ICU patients without thrombosis (n = 18). CONCLUSIONS: Our results suggest that severe COVID-19 is associated with a changed fibrin network structure and decreased susceptibility to fibrinolysis. Because these changes were not exclusive to COVID-19 patients, they may not explain the increased thrombosis risk.

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.

Inflammation/coagulopathy/fibrinolysis: Dynamic indicators of COVID-19 progression in patients with moderate COVID-19 in Wenzhou, China.

BACKGROUND: The majority of the coronavirus disease 2019 (COVID-19) non-survivors meet the criteria for disseminated intravascular coagulation (DIC). Although timely monitoring of clotting hemorrhagic development during the natural course of COVID-19 is critical for understanding pathogenesis, diagnosis, and treatment of the disease, however, limited data are available on the dynamic processes of inflammation/coagulopathy/fibrinolysis (ICF). METHODS: We monitored the dynamic progression of ICF in patients with moderate COVID-19. Out of 694 COVID-19 inpatients from 10 hospitals in Wenzhou, China, we selected 293 adult patients without comorbidities. These patients were divided into different daily cohorts according to the COVID-19 onset-time. Furthermore, data of 223 COVID-19 patients with comorbidities and 22 critical cases were analyzed. Retrospective data were extracted from electronic medical records. RESULTS: The virus-induced damages to pre-hospitalization patients triggered two ICF fluctuations during the 14-day course of the disease. C-reactive protein (CRP), fibrinogen, and D-dimer levels increased and peaked at day 5 (D) 5 and D9 during the 1st and 2nd fluctuations, respectively. The ICF activities were higher during the 2nd fluctuation. Although 12-day medication returned high CRP concentrations to normal and blocked fibrinogen increase, the D-dimer levels remained high on days 17 ±â€¯2 and 23 ±â€¯2 days of the COVID-19 course. Notably, although the oxygenation index, prothrombin time and activated partial thromboplastin time were within the normal range in critical COVID-19 patients at administration, 86% of these patients had a D-dimer level > 500 µg/L. CONCLUSION: COVID-19 is linked with chronic DIC, which could be responsible for the progression of the disease. Understanding and monitoring ICF progression during COVID-19 can help clinicians in identifying the stage of the disease quickly and accurately and administering suitable treatment.

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.

Endogenous fibrinolysis-Relevance to clinical thrombosis risk assessment.

The development of an obstructive luminal thrombus is pathological and considered a failure of endogenous fibrinolysis. The consequences may be fatal, or result in lasting downstream organ damage. Therefore, assessment of endogenous fibrinolytic status in an individual may identify those at risk of occlusive thrombus formation and provide prognostic information. Arterial thrombi are more platelet rich and more resistant to fibrinolysis than venous thrombi. Several recent studies using global tests of fibrinolysis in patients with acute coronary syndromes (ACS) have shown that despite dual antiplatelet therapy, patients with impaired fibrinolytic status have an increased risk of adverse cardiovascular events, compared with those with effective fibrinolytic function. Such data add significantly to the predictive value of established cardiovascular risk factors and conventional biomarkers. Most data reported have been obtained with the Global Thrombosis Test and the turbidimetric plasma clot lysis assay. A few small studies in patients with ischaemic stroke suggest a similar predictive role of fibrinolytic status assessment in these patients. Studies reporting an association between impaired fibrinolysis and future venous thrombotic events are limited, and in the form of case-control studies. Viscoelastic assays may have a role in the prediction of venous thromboembolic risk. Assays of fibrinolytic function should be used to obtain a more accurate risk of future thrombotic events, particularly in the setting of ACS. The availability of point-of-care tests helps facilitate this and should encourage future studies to assess personalised antithrombotic treatment combinations to optimise fibrinolytic status and reduce thrombosis risk.

Coagulation profile of COVID-19 patients admitted to the ICU: An exploratory study.

BACKGROUND: Coagulation abnormalities in COVID-19 patients have not been addressed in depth. OBJECTIVE: To perform a longitudinal evaluation of coagulation profile of patients admitted to the ICU with COVID-19. METHODS: Conventional coagulation tests, rotational thromboelastometry (ROTEM), platelet function, fibrinolysis, antithrombin, protein C and S were measured at days 0, 1, 3, 7 and 14. Based on median total maximum SOFA score, patients were divided in two groups: SOFA ≤ 10 and SOFA > 10. RESULTS: Thirty patients were studied. Some conventional coagulation tests, as aPTT, PT and INR remained unchanged during the study period, while alterations on others coagulation laboratory tests were detected. Fibrinogen levels were increased in both groups. ROTEM maximum clot firmness increased in both groups from Day 0 to Day 14. Moreover, ROTEM-FIBTEM maximum clot firmness was high in both groups, with a slight decrease from day 0 to day 14 in group SOFA ≤ 10 and a slight increase during the same period in group SOFA > 10. Fibrinolysis was low and decreased over time in all groups, with the most pronounced decrease observed in INTEM maximum lysis in group SOFA > 10. Also, D-dimer plasma levels were higher than normal reference range in both groups and free protein S plasma levels were low in both groups at baseline and increased over time, Finally, patients in group SOFA > 10 had lower plasminogen levels and Protein C ​​than patients with SOFA <10, which may represent less fibrinolysis activity during a state of hypercoagulability. CONCLUSION: COVID-19 patients have a pronounced hypercoagulability state, characterized by impaired endogenous anticoagulation and decreased fibrinolysis. The magnitude of coagulation abnormalities seems to correlate with the severity of organ dysfunction. The hypercoagulability state of COVID-19 patients was not only detected by ROTEM but it much more complex, where changes were observed on the fibrinolytic and endogenous anticoagulation system.

Impaired fibrinolysis in critically ill COVID-19 patients.

BACKGROUND: Critically ill coronavirus disease 2019 (COVID-19) patients present with a hypercoagulable state with high rates of macrovascular and microvascular thrombosis, for which hypofibrinolysis might be an important contributing factor. METHODS: We retrospectively analysed 20 critically ill COVID-19 patients at Innsbruck Medical University Hospital whose coagulation function was tested with ClotPro® and compared with that of 60 healthy individuals at Augsburg University Clinic. ClotPro is a viscoelastic whole blood coagulation testing device. It includes the TPA test, which uses tissue factor (TF)-activated whole blood with added recombinant tissue-derived plasminogen activator (r-tPA) to induce fibrinolysis. For this purpose, the lysis time (LT) is measured as the time from when maximum clot firmness (MCF) is reached until MCF falls by 50%. We compared COVID-19 patients with prolonged LT in the TPA test and those with normal LT. RESULTS: Critically ill COVID-19 patients showed hypercoagulability in ClotPro assays. MCF was higher in the EX test (TF-activated assay), IN test (ellagic acid-activated assay), and FIB test (functional fibrinogen assay) with decreased maximum lysis (ML) in the EX test (hypofibrinolysis) and highly prolonged TPA test LT (decreased fibrinolytic response), as compared with healthy persons. COVID-19 patients with decreased fibrinolytic response showed higher fibrinogen levels, higher thrombocyte count, higher C-reactive protein levels, and decreased ML in the EX test and IN test. CONCLUSION: Critically ill COVID-19 patients have impaired fibrinolysis. This hypofibrinolytic state could be at least partially dependent on a decreased fibrinolytic response.

Thromboembolic complications in critically ill COVID-19 patients are associated with impaired fibrinolysis.

BACKGROUND: There is emerging evidence for enhanced blood coagulation in coronavirus 2019 (COVID-19) patients, with thromboembolic complications contributing to morbidity and mortality. The mechanisms underlying this prothrombotic state remain enigmatic. Further data to guide anticoagulation strategies are urgently required. METHODS: We used viscoelastic rotational thromboelastometry (ROTEM) in a single-center cohort of 40 critically ill COVID-19 patients. RESULTS: Clear signs of a hypercoagulable state due to severe hypofibrinolysis were found. Maximum lysis, especially following stimulation of the extrinsic coagulation system, was inversely associated with an enhanced risk of thromboembolic complications. Combining values for maximum lysis with D-dimer concentrations revealed high sensitivity and specificity of thromboembolic risk prediction. CONCLUSIONS: The study identifies a reduction in fibrinolysis as an important mechanism in COVID-19-associated coagulopathy. The combination of ROTEM and D-dimer concentrations may prove valuable in identifying patients requiring higher intensity anticoagulation.

COVID-19 Associated Hypercoagulability: Manifestations, Mechanisms, and Management.

ABSTRACT: Patients with severe coronavirus disease-2019 (COVID-19) frequently have hypercoagulability caused by the immune response to the severe acute respiratory syndrome coronavirus-2 infection. The pathophysiology of COVID-19 associated hypercoagulability is not fully understood, but characteristic changes include: increased fibrinogen concentration, increased Factor VIII activity, increased circulating von Willebrand factor, and exhausted fibrinolysis. Anticoagulant therapy improves outcomes in mechanically ventilated patients with COVID-19 and viscoelastic coagulation testing offers an opportunity to tailor anticoagulant therapy based on an individual patient's coagulation status. In this narrative review, we summarize clinical manifestations of COVID-19, mechanisms, monitoring considerations, and anticoagulant therapy. We also review unique considerations for COVID-19 patients who are on extracorporeal membrane oxygenation.

COVID-19: The crucial role of blood coagulation and fibrinolysis.

The overflow of studies in the recent literature on COVID-19 often gives provisional or contradictory results and therefore deserves pauses of reflection and reconsideration. In fact, knowledges of pathophysiology of this new disease are still in development and hence originate discussions and interpretations. Regarding the role of blood coagulation and fibrinolysis, these mechanisms should be considered as crucial especially in severe cases. It is proposed to consider two distinct phenotypes of thrombotic manifestations: the current "thromboembolic type" also occurring in other kinds of sepsis, and the diffuse micro-thrombotic type, prevailing in the lungs but sometimes extending to other organs. Both types can induce severe disease and are potentially lethal. The micro-thrombotic pattern, more specific for COVID-19, results from a massive activation of coagulation strictly coupled with a hyper-intense inflammatory and immune reaction. This results in widespread occlusive thrombotic micro-angiopathy with destruction of alveoli and obstructive neoangiogenesis. The involvement of fibrinolysis, often neglected, confers a double faceted process of activation/inhibition, finally conducive to a fibrinolytic shutdown that reinforces persistence of micro-thrombi. Considering these peculiar mechanisms, it seems evident that both prophylactic and therapeutic effects of current anti-thrombotic drugs cannot be taken for granted and need therefore new specific and rigorous controlled trials.