ABSTRACT
OBJECTIVE: Granzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-ß1 into the extracellular milieu. METHODS/RESULTS: Three extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-ß1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-ß1 release. Our data confirmed that GrB liberated TGF-ß1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-ß1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells. CONCLUSION: In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-ß1 from PGs.
Subject(s)
Biglycan/metabolism , Decorin/metabolism , Granzymes/metabolism , Proteoglycans/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1/metabolism , Biocatalysis/drug effects , Blotting, Western , Cells, Cultured , Coumarins/pharmacology , Extracellular Matrix/metabolism , Extracellular Space/metabolism , Granzymes/antagonists & inhibitors , Humans , Isocoumarins , Kinetics , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Phosphorylation/drug effects , Proteoglycans/chemistry , Receptors, Transforming Growth Factor beta/chemistry , Serine Proteinase Inhibitors/pharmacology , Smad3 Protein/metabolism , Solubility , Substrate Specificity , Transforming Growth Factor beta1/pharmacologyABSTRACT
The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.
Subject(s)
Anti-Bacterial Agents/pharmacology , Blood Coagulation/drug effects , Inflammation/pathology , Metal Nanoparticles/toxicity , Silver/pharmacology , Silver/toxicity , Bacteria/drug effects , Cell Death/drug effects , Cell Line , Cytokines/metabolism , Humans , Inflammation Mediators/metabolism , Light , Macrophages/cytology , Macrophages/drug effects , Metal Nanoparticles/ultrastructure , Microbial Sensitivity Tests , Particle Size , Scattering, RadiationABSTRACT
Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain.
