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.

Comparison of the coagulopathies associated with COVID-19 and sepsis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with activation of coagulation that mainly presents as thrombosis. Sepsis is also associated with activation of coagulation that mainly presents as disseminated intravascular coagulation. Many studies have reported increased levels of plasma d-dimer in patients with COVID-19 that is associated with severity, thrombosis, and mortality. OBJECTIVES: The aim of this study was to compare levels of circulating extracellular vesicle tissue factor (EVTF) activity and active plasminogen activator inhibitor 1 (PAI-1) in plasma from patients with COVID-19 or sepsis. METHODS: We measured levels of d-dimer, EVTF activity, and active PAI-1 in plasma samples from patients with COVID-19 (intensive care unit [ICU], N = 15; and non-ICU, N = 20) and patients with sepsis (N = 35). RESULTS: Patients with COVID-19 had significantly higher levels of d-dimer, EVTF activity, and active PAI-1 compared with healthy controls. Patients with sepsis had significantly higher levels of d-dimer and EVTF activity compared with healthy controls. Levels of d-dimer were significantly lower in patients with COVID-19 compared with patients with sepsis. Levels of EVTF activity were significantly higher in ICU patients with COVID-19 compared with patients with sepsis. Levels of active PAI-1 were significantly higher in patients with COVID-19 compared with patients with sepsis. CONCLUSIONS: High levels of both EVTF activity and active PAI-1 may promote thrombosis in patients with COVID-19 due to simultaneous activation of coagulation and inhibition of fibrinolysis. The high levels of active PAI-1 in patients with COVID-19 may limit plasmin degradation of crosslinked fibrin and the release of d-dimer. This may explain the lower levels of D-dimer in patients with COVID-19 compared with patients with sepsis.

COVID-19 pandemic perspectives: A scientific silver lining?

Accounts of the numerous negative effects caused by COVID-19 are pervasive, but few perspectives have identified any positive impacts of this massive societal shift. This forum examines potentially positive changes that have occurred within the scientific community amid the chaotic pandemic. Among these positives are the formation of virtual supergroups and an interdisciplinary brain trust. In forcing scientists away from their lab benches, COVID-19 has created time and space for more conversations about science and experimental design. Being away from the lab in this time of social unrest has also given scientists time to directly address institutional racism and its suppression of diversity in science. Although COVID-19 has been an unforeseen disaster of epic proportions, some of the resulting changes in our scientific community should remain in place after the pandemic is over. By leveraging these small wins, we will undoubtedly return to our laboratories stronger, smarter, and more efficient.

Fibrinogen, Fibrin, and Fibrin Degradation Products in COVID-19.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the highly pathogenic and highly transmissible human coronavirus that is the causative agent for the worldwide COVID-19 pandemic. COVID-19 manifests predominantly as a respiratory illness with symptoms consistent with viral pneumonia, but other organ systems (e.g., kidney, heart, brain) can also become perturbed in COVID-19 patients. Accumulating data suggest that significant activation of the hemostatic system is a common pathological manifestation of SARS-CoV-2 infection. The clotting protein fibrinogen is one of the most abundant plasma proteins. Following activation of coagulation, the central coagulation protease thrombin converts fibrinogen to fibrin monomers, which selfassemble to form a matrix, the primary structural component of the blood clot. Severe COVID-19 is associated with a profound perturbation of circulating fibrinogen, intra- and extravascular fibrin deposition and persistence, and fibrin degradation. Current findings suggest high levels of fibrinogen and the fibrin degradation product D-dimer are biomarkers of poor prognosis in COVID-19. Moreover, emerging studies with in vitro and animal models indicate fibrin(ogen) as an active player in COVID-19 pathogenesis. Here, we review the current literature regarding fibrin(ogen) and COVID-19, including possible pathogenic mechanisms and treatment strategies centered on clotting and fibrin(ogen) function.

Understanding COVID-19-associated coagulopathy.

COVID-19-associated coagulopathy (CAC) is a life-threatening complication of SARS-CoV-2 infection. However, the underlying cellular and molecular mechanisms driving this condition are unclear. Evidence supports the concept that CAC involves complex interactions between the innate immune response, the coagulation and fibrinolytic pathways, and the vascular endothelium, resulting in a procoagulant condition. Understanding of the pathogenesis of this condition at the genomic, molecular and cellular levels is needed in order to mitigate thrombosis formation in at-risk patients. In this Perspective, we categorize our current understanding of CAC into three main pathological mechanisms: first, vascular endothelial cell dysfunction; second, a hyper-inflammatory immune response; and last, hypercoagulability. Furthermore, we pose key questions and identify research gaps that need to be addressed to better understand CAC, facilitate improved diagnostics and aid in therapeutic development. Finally, we consider the suitability of different animal models to study CAC.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Assessing Plasmin Generation in Health and Disease.

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.

SARS-CoV-2 suppresses anticoagulant and fibrinolytic gene expression in the lung.

Extensive fibrin deposition in the lungs and altered levels of circulating blood coagulation proteins in COVID-19 patients imply local derangement of pathways that limit fibrin formation and/or promote its clearance. We examined transcriptional profiles of bronchoalveolar lavage fluid (BALF) samples to identify molecular mechanisms underlying these coagulopathies. mRNA levels for regulators of the kallikrein-kinin (C1-inhibitor), coagulation (thrombomodulin, endothelial protein C receptor), and fibrinolytic (urokinase and urokinase receptor) pathways were significantly reduced in COVID-19 patients. While transcripts for several coagulation proteins were increased, those encoding tissue factor, the protein that initiates coagulation and whose expression is frequently increased in inflammatory disorders, were not increased in BALF from COVID-19 patients. Our analysis implicates enhanced propagation of coagulation and decreased fibrinolysis as drivers of the coagulopathy in the lungs of COVID-19 patients.

COVID-19 and Sepsis Are Associated With Different Abnormalities in Plasma Procoagulant and Fibrinolytic Activity.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of sepsis. However, clinical laboratory metrics suggest key differences in these pathologies. We sought to determine whether plasma coagulation and fibrinolytic potential in patients with COVID-19 differ compared with healthy donors and critically ill patients with sepsis. Approach and Results: We performed comparative studies on plasmas from a single-center, cross-sectional observational study of 99 hospitalized patients (46 with COVID-19 and 53 with sepsis) and 18 healthy donors. We measured biomarkers of endogenous coagulation and fibrinolytic activity by immunoassays, thrombin, and plasmin generation potential by fluorescence and fibrin formation and lysis by turbidity. Compared with healthy donors, patients with COVID-19 or sepsis both had elevated fibrinogen, d-dimer, soluble TM (thrombomodulin), and plasmin-antiplasmin complexes. Patients with COVID-19 had increased thrombin generation potential despite prophylactic anticoagulation, whereas patients with sepsis did not. Plasma from patients with COVID-19 also had increased endogenous plasmin potential, whereas patients with sepsis showed delayed plasmin generation. The collective perturbations in plasma thrombin and plasmin generation permitted enhanced fibrin formation in both COVID-19 and sepsis. Unexpectedly, the lag times to thrombin, plasmin, and fibrin formation were prolonged with increased disease severity in COVID-19, suggesting a loss of coagulation-initiating mechanisms accompanies severe COVID-19. CONCLUSIONS: Both COVID-19 and sepsis are associated with endogenous activation of coagulation and fibrinolysis, but these diseases differently impact plasma procoagulant and fibrinolytic potential. Dysregulation of procoagulant and fibrinolytic pathways may uniquely contribute to the pathophysiology of COVID-19 and sepsis.

Coagulation Abnormalities and Thrombosis in Patients Infected With SARS-CoV-2 and Other Pandemic Viruses.

The world is amid a pandemic caused by severe acute respiratory syndrome-coronavirus 2. Severe acute respiratory syndrome-coronavirus causes serious respiratory tract infections that can lead to viral pneumonia, acute respiratory distress syndrome, and death. Some patients with coronavirus disease 2019 (COVID-19) have an activated coagulation system characterized by elevated plasma levels of d-dimer-a biomarker of fibrin degradation. Importantly, high levels of D-dimer on hospital admission are associated with increased risk of mortality. Venous thromboembolism is more common than arterial thromboembolism in hospitalized COVID-19 patients. Pulmonary thrombosis and microvascular thrombosis are observed in autopsy studies, and this may contribute to the severe hypoxia observed in COVID-19 patients. It is likely that multiple systems contribute to thrombosis in COVID-19 patients, such as activation of coagulation, platelet activation, hypofibrinolysis, endothelial cell dysfunction, inflammation, neutrophil extracellular traps, and complement. Targeting these different pathways may reduce thrombosis and improve lung function in COVID-19 patients.