- Molecular mechanisms of fibrinolysis
- Modulation of serpin-fibrinolysin interactions in vivo
- Fibrinolytic therapy in infectious, loculating animal models of pleural injury
- Novel assays for evaluating disease severity and predicting therapeutic outcomes
- Design and characterization of new inhibitors of PAI-1
Impaired fibrinolysis results in fibrin accumulation, and causes a number of serious illnesses, many of which have high morbidity and mortality. Vascular occlusion due to the pathological accumulation of blood components (platelets, fibrinogen, clotting factors, etc.) in the circulation results in thrombotic disease, the number one killer in the industrialized nations. Extravascular fibrosis in the respiratory system and pleural space is often associated with high mortality, resistance to conservative therapy. Both the incidence and high mortality (up to 20%) from empyema and pleural sepsis have not decreased, despite the latest developments in medicine, and costs of up to $0.5B annually. We believe that the current therapeutic approaches to treating fibrosis could be improved using our understanding of the fundamental molecular mechanisms of fibrinolysis. Recently, we validated active plasminogen activator inhibitor 1 (PAI-1) as a biomarker and molecular target in an animal model of pleural injury. PAI-1 neutralization resulted in an 8-fold increase in the efficacy of fibrinolytic therapy in our model. Currently, we are developing novel, less invasive, and safer PAI-1 targeting fibrinolytic therapies and diagnostic assays, so we can provide safer, more effective treatments for patients with pleural loculation and lung injury.
Our main research interest is the evaluation of the fundamental mechanisms that govern the molecular interactions in the fibrinolytic system, and their application to translational research (bench-to-bedside via animal models), focused on increasing the efficacy and safety of fibrinolytic therapy. Four interconnected projects (I-IV) form the core of these studies:
I. The study of the molecular mechanisms governing fibrinolysis and intermolecular mechanisms that modulate serpin-fibrinolysin interactions. We have shown that the relatively slow rate of intrapleural fibrinolysis, the half-life of the fibrinolysin, and elevated levels of active PAI-1 dramatically affect the outcomes of fibrinolytic therapy in pleural injury. Current studies include evaluation of the effects of: (i) nucleic acids on fibrin structure and fibrinolysis; (ii) serpins’ contribution to the development of pleural and lung injury and to the outcomes of fibrinolytic therapy; (iii) mechanisms of stabilization of PAI-1 activity in vivo; (iv) mechanisms of modulation of endogenous PAI-1 activity via intermolecular interactions.
II. Fibrinolytic therapy of pleural injury in infectious and loculating animal models. Recently, we validated that active PAI-1 is a biomarker and molecular target for fibrinolytic therapy in an animal model of pleural fibrosis. Targeting active PAI-1 increases the efficacy of fibrinolytic therapy in the animal model (decreasing the dose of plasminogen activator needed for effective treatment of pleural fibrosis) up to 8-fold. Several intermolecular mechanisms of PAI-1 neutralization previously characterized in vitro have been tested in vivo to determine the contribution of active PAI-1 to the efficacy of fibrinolytic therapy. Mechanisms of fibrinolysin processing in vivo and evaluation of the optimal strategies for PAI-1 neutralization in infectious and loculating animal models of pleural injury are currently under investigation.
III. Developing novel assays for evaluating of the severity of pleural injury ex vivo and predicting possible outcomes of fibrinolytic therapy. Our data demonstrate that, while the basic mechanisms governing fibrinolysis during treatment are similar, the different levels of key proteins which differ patient to patient could dramatically affect the outcomes of fibrinolytic therapy. This project aims to develop a new companion assay to personalize fibrinolytic therapy based on the ex vivo analysis of a pattern of molecular signatures in biological samples.
IV. The design and characterization of new PAI-1 inhibitors. At present, monoclonal antibodies (mAbs) are the most specific and selective inhibitors of PAI-1. However, therapeutic use favors the search for small (low molecular weight, LMW) molecules that inhibit PAI-1 with similar efficacy. Our fundamental knowledge of serpin mechanisms has built a foundation for the development and assessment of new PAI-1 inhibitors. Known mechanisms of PAI-1 inactivation are employed for (i) the selection of peptides specific for the epitopes of PAI-1 neutralizing mAbs that could be used in combination, (ii) studies of the additivity between different intermolecular mechanisms affecting PAI-1 activity, (iii) further minimization of PAI-1 specific ligands: mAb (150kDa) → Fab (40kDa) → scFv (20kDa) → peptide (1-2kDa) → LMW inhibitor (0.5-1.5kDa), and (iv) the development and assessment of new multivalent modulators of PAI-1.
Our goals are to understand the molecular mechanisms governing fibrinolysis, develop novel approaches to personalize fibrinolytic therapy and predict outcomes based on the analysis of biological samples. PAI-1, a biomarker and molecular target in pleural injury, is a serpin that plays an important role in the control of normal and pathological thrombosis (clotting) and fibrinolysis (clot breakdown). PAI-1 is a mechanism-based inhibitor of urokinase (uPA) and tissue type (tPA) plasminogen activators. PAI-1 prevents the formation of plasmin, thus inhibiting the fibrinolytic system and promoting thrombosis in the vasculature and in extravascular fibrin deposition seen in acute lung and pleural injury, cystic fibrosis, etc. High levels of PAI-1 are associated with a number of life threatening conditions such as myocardial infarction (heart attacks), sepsis (blood poisoning), atherosclerosis, angina pectoris, pleural and acute lung injury and correlate with unfavorable outcomes in disseminated intravascular coagulation (DIC, sepsis), acute lung and pleural injury, and in a number of cancers. PAI-1 neutralization resulted in 8-fold increase in the efficacy of fibrinolytic therapy in our animal model of pleural injury. Analysis of human samples supports the idea that there is interspecies similarity of the mechanisms governing fibrinolysis in pleural injury, and, thus, the possible critical role of active PAI-1 in disease severity and predicting disease outcomes. While the general mechanisms are similar, the pattern of molecular signatures that form the “footprint” of the disease is unique for each patient. We believe that decoding these molecular signatures is the key for developing personalized fibrinolytic therapy, which would provide maximal benefits for each individual patient and increase the probability of successful therapy with minimized risk for complications.
Florova G, Azghani A, Karandashova S, Schaefer C, Koenig K, Stewart-Evans K, Declerck PJ, Idell S, Komissarov AA. Targeting of Plasminogen Activator Inhibitor 1 Improves Fibrinolytic Therapy for Tetracycline Induced Pleural Injury in Rabbits. Am J Respir Cell Mol Biol. 2015 Apr;52(4):429-37.PMID:25140386
Ji HL, Zhao R, Komissarov AA, Chang Y, Liu Y, Matthay MA. HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/25555911" Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites. J Biol Chem. 2015 Feb 27;290(9):5241-55. PMID:25555911
Komissarov AA, Florova G, Azghani A, Karandashova S, Kurdowska AK, Idell S. Active α-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids. Am J Physiol Lung Cell Mol Physiol. 2013 Nov 15;305(10):L682-92 PMID: 23997178
Florova G, Karandashova S, Declerck PJ, Idell S, Komissarov AA. Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex. Biochemistry. 2013 Jul 9;52(27):4697-709. PMID: 23734661
Karandashova S, Florova G, Azghani AO, Komissarov AA, Koenig K, Tucker TA, Allen TC, Stewart K, Tvinnereim A, Idell S. Intrapleural adenoviral delivery of human plasminogen activator inhibitor-1 exacerbates tetracycline-induced pleural injury in rabbits. Am J Respir Cell Mol Biol. 2013 Jan; 48(1):44-52. PMID: 23002099
Tucker TA, Jeffers A, Alvarez A, Owens S, Koenig K, Quaid B, Komissarov AA, Florova G, Kothari H, Pendurthi U, Rao LV, Idell S. Plasminogen Activator Inhibitor-1 Deficiency Augments Visceral Mesothelial Organization, Intrapleural Coagulation and Lung Restriction in Mice with Carbon Black/Bleomycin-Induced Pleural Injury. Am J Respir Cell Mol Biol. 2013 Sep 11. [Epub ahead of print] PMID: 24024554
Komissarov AA, Stankowska D, Krupa A, Fudala R, Florova G, Florence J, Fol M, Allen TC, Idell S, Matthay MA, Kurdowska AK. Novel aspects of urokinase function in the injured lung: role of α2-macroglobulin. Am J Physiol Lung Cell Mol Physiol. 2012 Dec 15;303(12):L1037-45. doi: 10.1152/ajplung.00117.2012. PMID: 23064953
Komissarov AA, Florova G, Idell S. Effects of extracellular DNA on plasminogen activation and fibrinolysis. J Biol Chem. 2011 Dec 9;286(49):41949-62. PMID: 21976662