Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.     

Blood/plasma secretome and microvesicles

A major but hitherto overseen component of the blood/plasma secretome is that of extracellular vesicles (EVs) which are shed from all blood cell types. These EVs are made up of microvesicles (MVs) and exosomes. MVs, 100 nm–1 μm in diameter, are released from the cell surface, and are a rich source of non-conventionally secreted proteins lacking a conventional signal peptide, and thus not secreted by the classical secretory pathways. Exosomes are smaller vesicles (≤ 100 nm) having an endocytic origin and released upon multivesicular body fusion with the plasma membrane. Both vesicle types play major roles in intercellular cross talk and constitute an important component of the secretome especially in the area of biomarkers for cancer. The release of EVs, which are found in all the bodily fluids, is enhanced in cancer and a major focus of cancer proteomics is therefore targeted at EVs. The blood/plasma secretome is also a source of EVs, potentially diagnostic of infectious disease, whether from EVs released from infected cells or from the pathogens themselves. Despite the great excitement in this field, as is stated here and in other parts of this Special issue entitled: An Updated Secretome, much of the EV research, whether proteomic or functional in nature, urgently needs standardisation both in terms of nomenclature and isolation protocols. This article is part of a Special Issue entitled: An Updated Secretome.     

Ethyl gallate, a scavenger of hydrogen peroxide that inhibits lysozyme-induced hydrogen peroxide signaling in vitro, reverses hypotension in canine septic shock

Although hydrogen peroxide (H2O2) is a well-described reactive oxygen species that is known for its cytotoxic effects and associated tissue injury, H2O2 has recently been established as an important signaling molecule. We previously demonstrated that lysozyme (Lzm-S), a mediator of sepsis that is released from leukocytes, could produce vasodilation in a phenylephrine-constricted carotid artery preparation by H2O2 signaling. We found that Lzm-S could intrinsically generate H2O2 and that this generation activated H2O2-dependent pathways. In the present study, we used this carotid artery preparation as a bioassay to define those antioxidants that could inhibit Lzm-S's vasodilatory effect. We then determined whether this antioxidant could reverse the hypotension that developed in an Escherichia coli bacteremic model. Of the many antioxidants tested, we found that ethyl gallate (EG), a nonflavonoid phenolic compound, was favorable in inhibiting Lzm-S-induced vasodilation. In our E. coli model, we found that EG reversed the hypotension that developed in this model and attenuated end-organ dysfunction. By fluorometric H2O2 assay and electrochemical probe techniques, we showed that EG could scavenge H2O2 and that it could reduce H2O2 production in model systems. These results show that EG, an antioxidant that was found to scavenge H2O2 in vitro, was able to attenuate cardiovascular dysfunction in a canine in vivo preparation. Antioxidants such as EG may be useful in the treatment of hemodynamic deterioration in septic shock.     

Lysozyme, a mediator of sepsis, impairs the cardiac neural adrenergic response by nonendothelial release of NO and inhibitory G protein signaling

We previously showed that lysozyme (Lzm-S), derived from leukocytes, caused myocardial depression in canine sepsis by binding to the endocardial endothelium to release nitric oxide (NO). NO then diffuses to adjacent myocytes to activate the cGMP pathway. In a canine right ventricular trabecular (RVT) preparation, Lzm-S also decreased the inotropic response to field stimulation (FSR) during which the sympathetic and parasympathetic nerves were simulated to measure the adrenergic response. In the present study, we determined whether the pathway by which Lzm-S decreased FSR was different from the pathway by which Lzm-S reduced steady-state (SS) contraction. Furthermore, we determined whether the decrease in FSR was due to a decrease in sympathetic stimulation or enhanced parasympathetic signaling. In the RVT preparation, we found that the inhibitory effect of Lzm-S on FSR was prevented by NO synthase (NOS) inhibitors. A cGMP inhibitor also blocked the depressant activity of Lzm-S. However, in contrast to the Lzm-S-induced decline in SS contraction, chemical removal of the endocardial endothelium by Triton X-100 to eliminate endothelial NO release did not prevent the decrease in FSR. An inhibitory G protein was involved in the effect of Lzm-S, since FSR could be restored by treatment with pertussis toxin. Atropine prevented the Lzm-S-induced decline in FSR, whereas beta(1)- and beta(2)-adrenoceptor function was not impaired by Lzm-S. These results indicate that the Lzm-S-induced decrease in FSR results from a nonendothelial release of NO. NO then acts through inhibitory G protein to enhance parasympathetic signaling.