From Loop to Strand: Characterization of the Conformation and Dynamics of the Human Plasminogen Activator Inhibitor-1 Reactive Center

Plasminogen activator inhibitor-1 (PAI-1), with its cofactor vitronectin (VN), controls the rate of plasmin-mediated fibrin breakdown in blood clots by inhibiting tissue-plasminogen activator (tPA) and urokinase-plasminogen activator (uPA). The activity of PAI-1 is attributed to its reactive center loop (RCL), which is solvent-exposed in an active conformation, but inserts as an additional strand into its central β [beta]-sheet during transition to a latent state and during inhibition. VN slows the latency transition, and the rate at which PAI-1 inhibits the plasminogen activators (PAs) also differs. However, the steps during the latency transition, mechanism of VN stabilization, and basis for inhibitory rate differences are unclear, and all involve the RCL. To address these issues, this study combines computational methods with cysteine-scanning mutagenesis of the RCL for fluorescence and electron paramagnetic resonance (EPR) spectroscopy to investigate changes in the RCL due to interactions with these ligands. Homology modeling of the RCL indicates sampling of a limited energy-conformation landscape for this region. Fluorescence investigation of the latency transition suggest that RCL detachment to assume the latent conformation occurs within the first 10 minutes of the process, which typically has a half-life of about 1 hour. Equilibrium-binding studies indicate that VN, its N-terminal somatomedin B (SMB) domain, and a longer truncation involving an intrinsically disordered domain (SMB-IDD) increase the solvent exposure of the RCL in stabilizing PAI-1. Studies with active site-blocked PAs reveal that both dock at the RCL, but rest differently on its top, employing distinct exosite interactions and mobility constraints on the RCL that likely effect the kinetics of its interaction with PAI-1. Thereby, this study provides detailed structural information on the PAI-1 RCL, and new insights into the latency process and interaction with PAs. Such information is valuable in the development of inhibitors specific for the interaction of PAI-1 with either PA, and in targeting this biomarker in diseases states caused by the dysregulation of PAI-1. Overall, the results from this work reveal that ligand interactions fine-tune the activity of PAI-1 by affecting the conformation and dynamics of the RCL from its position as a solvent-exposed loop to an inserted β [beta]-strand

Studies on the Role of Vitronectin and Plasminogen-Activator Inhibitor-1 Complexes Beyond Inhibiting Proteases: Binding to the Extracellular Matrix, Cell Interactions and Pathogenesis

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in blood in a complex with vitronectin (VN). These two proteins are also found localized together in the extracellular matrix in many different pathophysiological conditions. Both of these proteins are involved with a number of physiologically important processes. Though PAI-1 is a well-known inhibitor of serine proteases, more emphasis is now geared towards its protease independent functions. VN, on the other hand, is a binding protein that exists in the circulation in a preferred monomeric conformation. However, in the extracellular matrix, VN exists as multimer with altered conformation. Though the exact reason for such conformational alterations and compartmentalization is unknown, there are a number of biomolecules, including PAI-1 that are proposed to cause such alterations. In last few years, sufficient experimental evidence has been gathered to confirm this protease- independent effect of PAI-1 by which it induces multimerization of VN in a concentration-dependent fashion. It has been observed also that PAI-1 remains associated with this multimeric complex for several hours. A major focus of this dissertation work was to extend our understanding of the mechanism of the interaction between these proteins and to explore the physiological relevance of the multimeric complexes formed by their interaction on cellular adhesion and migration. In our study, emphasis has been given to the presence of an appropriate microenvironment so that the role of the multimeric complexes could be investigated in a relevant biological setting. Our findings indicate the importance of the surrounding microenvironment in establishing the specific role of the VN/PAI-1 complex in cell-matrix interactions. In a previous study from our lab, it was found that vitronectin knock-out mice were more resistant to Candida infection compared to wild type C57Bl/6 mice. One of the goals of this dissertation work was to provide a mechanistic explanation for their increased survival of the vitronectin knock-out mice upon Candida infection. Another important aspect of this work was to establish biophysical methods for understanding the structural changes that happen in PAI-1 naturally or due to ligand binding

A local uPAR-plasmin-TGFβ1 positive feedback loop in a qualitative computational model of angiogenic sprouting explains the in vitro effect of fibrinogen variants

In experimental assays of angiogenesis in three-dimensional fibrin matrices, a temporary scaffold formed during wound healing, the type and composition of fibrin impacts the level of sprouting. More sprouts form on high molecular weight (HMW) than on low molecular weight (LMW) fibrin. It is unclear what mechanisms regulate the number and the positions of the vascular-like structures in cell cultures. To address this question, we propose a mechanistic simulation model of endothelial cell migration and fibrin proteolysis by the plasmin system. The model is a hybrid, cell-based and continuum, computational model based on the cellular Potts model and sets of partial-differential equations. Based on the model results, we propose that a positive feedback mechanism between uPAR, plasmin and transforming growth factor β1 (TGFβ1) selects cells in the monolayer for matrix invasion. Invading cells releases TGFβ1 from the extracellular matrix through plasmin-mediated fibrin degradation. The activated TGFβ1 further stimulates fibrin degradation and keeps proteolysis active as the sprout invades the fibrin matrix. The binding capacity for TGFβ1 of LMW is reduced relative to that of HMW. This leads to reduced activation of proteolysis and, consequently, reduced cell ingrowth in LMW fibrin compared to HMW fibrin. Thus our model predicts that endothelial cells in LMW fibrin matrices compared to HMW matrices show reduced sprouting due to a lower bio-availability of TGFβ1

ANALYSIS OF THE NETWORK DYNAMICS OF TRANSFORMING GROWTH FACTOR - ?1 BISTABLE ACTIVATION, WITH IMPLICATIONS ON BISTABILITY AND COMBINATION THERAPY

Ph.DDOCTOR OF PHILOSOPH

Proteases of the neutrophil membrane represent an alternative fibrinolytic pathway to that mediated by plasmin

The cellular components of the blood, which become associated with fibrin through specific cellular adhesive processes, play a significant role in the breakdown of fibrin. Fibrinolysis by elastase and cathepsin G, enzymes present within the azurophilic granules of the neutrophil, has previously been shown. Recent studies have demonstrated neutrophil-mediated fibrinogenolysis by a membrane-associated protease which suggests that proteases connected with the neutrophil membrane might also be capable of clot dissolution. Investigations showed that neutrophil-mediated clot lysis was effected by a membrane-associated serine protease that can be dissociated by SDS-PAGE to bands that migrate to apparent molecular weights of 501 kDa, 398 kDa, 316 kDa, 245 kDa and 209 kDa. This degradation was distinct from that produced by plasmin, neutrophil lysosomal enzymes and purified human neutrophil elastase and enhanced the action of plasmin in clot solubilization. Preincubation of neutrophils with monoclonal antibodies directed against the CD 11 c/CD 18 integrin was able to significantly inhibit neutrophil membrane-dependent fibrinolytic activity. Upregulation of enzyme activity occurred following association of fibrin substrate with the cell membrane and was dependent on the activation of cellular kinases, in particular protein kinase C. Fibrin products generated by neutrophil membrane proteolytic activity were found to possess biological activity. The low molecular weight peptides effected substantial inhibition of thrombin-induced platelet aggregation while the presence of the higher molecular weight material could partially overcome platelet-induced resistance to plasmic lysis. No modulation of platelet-mediated fibrin clot retraction was observed using these same fibrin products. Neutrophil lysosomal enzyme activity was shown to further degrade the end products of plasmic fibrin degradation into low molecular weight material, followed by reassembly of higher molecular weight products in a process dependent on calcium and factor XIII. The reformed products have a similar molecular weight to those produced by plasmic lysis of fibrin, as well as a putative crosslinked site. However, the isoelectric point of these reformed products indicates they are distinctly different from plasmin-derived fibrin products. These reassembled products were recognized by a monoclonal antibody raised against D-dimer. Processing by neutrophils of the end products of plasmic fibrin degradation may have the potential for modulating the immune response as well as compromising the predictive value of tests measuring D-dimer, used as a laboratory marker of a number of thromboembolic disorders encountered in clinical practice

On Quantitative Comparison of Chemical Reaction Network Models

Chemical reaction networks (CRNs) provide a convenient language for modelling a broad variety of biological systems. These models are commonly studied with respect to the time series they generate in deterministic or stochastic simulations. Their dynamic behaviours are then analysed, often by using deterministic methods based on differential equations with a focus on the steady states. Here, we propose a method for comparing CRNs with respect to their behaviour in stochastic simulations. Our method is based on using the flux graphs that are delivered by stochastic simulations as abstract representations of their dynamic behaviour. This allows us to compare the behaviour of any two CRNs for any time interval, and define a notion of equivalence on them that overlaps with graph isomorphism at the lowest level of representation. The similarity between the compared CRNs can be quantified in terms of their distance. The results can then be used to refine the models or to replace a larger model with a smaller one that produces the same behaviour or vice versa.Comment: In Proceedings HCVS/PERR 2019, arXiv:1907.0352

Mechanisms of Fibrosis in Regeneration of Aged Skeletal Muscle

Skeletal muscle is a highly adaptive tissue that possesses the ability to regenerate following damage. Regenerative capacity of skeletal muscle declines as age advances, leading to restricted mobility and poor quality of life. The impairment of the regeneration process in aged muscle is partly due to the accumulation of structural proteins in the extracellular matrix (ECM), termed fibrosis. In this study, two important proteins regulating ECM remodeling, plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-9 (MMP-9), were investigated to determine their role in modulating changes in the ECM during aged muscle regeneration. The regeneration process was studied in young (3 month old) and aged (18 month old) C56BL/6J mice at 3, 5, and 7 days following cardiotoxin-induced muscle damage. The regeneration process was significantly impaired in aged muscle as indicated by decreased muscle mass, cross-sectional fibre area, number of newly regenerated myofibres, and eMHC expression. Greater PAI-1 expression was found in aged regenerating myofibres 5 and 7 days following damage. Aged muscle displayed significantly greater extramyocellular PAI-1 acutely following damage, accumulation of collagen I, and delayed macrophage infiltration. Active MMP-9 was lower in aged muscle 3 and 5 days following damage compared to young muscle. However, MMP-9 primarily colocalized with F4/80+ macrophages in young muscle, but this colocalization was reduced in aged muscle. Taken together, this study provides a foundation for the mechanisms underlying the impairment of muscle regeneration in aged muscle