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.

The Role of the Plasminogen/Plasmin System in Inflammation of the Oral Cavity.

The oral cavity is a unique environment that consists of teeth surrounded by periodontal tissues, oral mucosae with minor salivary glands, and terminal parts of major salivary glands that open into the oral cavity. The cavity is constantly exposed to viral and microbial pathogens. Recent studies indicate that components of the plasminogen (Plg)/plasmin (Pm) system are expressed in tissues of the oral cavity, such as the salivary gland, and contribute to microbial infection and inflammation, such as periodontitis. The Plg/Pm system fulfills two major functions: (a) the destruction of fibrin deposits in the bloodstream or damaged tissues, a process called fibrinolysis, and (b) non-fibrinolytic actions that include the proteolytic modulation of proteins. One can observe both functions during inflammation. The virus that causes the coronavirus disease 2019 (COVID-19) exploits the fibrinolytic and non-fibrinolytic functions of the Plg/Pm system in the oral cavity. During COVID-19, well-established coagulopathy with the development of microthrombi requires constant activation of the fibrinolytic function. Furthermore, viral entry is modulated by receptors such as TMPRSS2, which is necessary in the oral cavity, leading to a derailed immune response that peaks in cytokine storm syndrome. This paper outlines the significance of the Plg/Pm system for infectious and inflammatory diseases that start in the oral cavity.

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.

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.

Structural analysis of experimental drugs binding to the SARS-CoV-2 target TMPRSS2.

The emergence of SARS-CoV-2 has prompted a worldwide health emergency. There is an urgent need for therapeutics, both through the repurposing of approved drugs and the development of new treatments. In addition to the viral drug targets, a number of human drug targets have been suggested. In theory, targeting human proteins should provide an advantage over targeting viral proteins in terms of drug resistance, which is commonly a problem in treating RNA viruses. This paper focuses on the human protein TMPRSS2, which supports coronavirus life cycles by cleaving viral spike proteins. The three-dimensional structure of TMPRSS2 is not known and so we have generated models of the TMPRSS2 in the apo state as well as in complex with a peptide substrate and putative inhibitors to aid future work. Importantly, many related human proteases have 80% or higher identity with TMPRSS2 in the S1-S1' subsites, with plasminogen and urokinase-type plasminogen activator (uPA) having 95% identity. We highlight 376 approved, investigational or experimental drugs targeting S1A serine proteases that may also inhibit TMPRSS2. Whilst the presence of a relatively uncommon lysine residue in the S2/S3 subsites means that some serine protease inhibitors will not inhibit TMPRSS2, this residue is likely to provide a handle for selective targeting in a focused drug discovery project. We discuss how experimental drugs targeting related serine proteases might be repurposed as TMPRSS2 inhibitors to treat coronaviruses.

Elevated Plasmin(ogen) as a Common Risk Factor for COVID-19 Susceptibility.

Patients with hypertension, diabetes, coronary heart disease, cerebrovascular illness, chronic obstructive pulmonary disease, and kidney dysfunction have worse clinical outcomes when infected with SARS-CoV-2, for unknown reasons. The purpose of this review is to summarize the evidence for the existence of elevated plasmin(ogen) in COVID-19 patients with these comorbid conditions. Plasmin, and other proteases, may cleave a newly inserted furin site in the S protein of SARS-CoV-2, extracellularly, which increases its infectivity and virulence. Hyperfibrinolysis associated with plasmin leads to elevated D-dimer in severe patients. The plasmin(ogen) system may prove a promising therapeutic target for combating COVID-19.