Exhaled Breath Condensate Pepsin: Potential Noninvasive Test for Gastroesophageal Reflux in COPD and Bronchiectasis.

BACKGROUND: Acid gastroesophageal reflux is a common problem in non-cystic fibrosis bronchiectasis and COPD. Invasive methods are used to diagnose gastroesophageal reflux, but the ability to detect pulmonary microaspiration of gastric contents using this method is unclear. A noninvasive option to detect pulmonary microaspiration is to measure pepsin in exhaled breath condensate (EBC), but this has not been related to esophageal pH monitoring in these lung conditions. This study aimed to measure pepsin concentrations and pH in EBC and to determine the relationship to gastroesophageal reflux in bronchiectasis or COPD. METHODS: Subjects with bronchiectasis (n=10) or COPD (n=10) and control subjects (n=10) completed 24-h esophageal pH monitoring for detection of acid gastroesophageal reflux, measuring the percentage of reflux time in the proximal esophagus and the DeMeester score (DMS). Concurrently, 3 samples of EBC were collected from each subject, and pH was measured and pepsin concentrations were analyzed by enzyme-linked immunosorbent assay. RESULTS: EBC pepsin was detected in subjects with bronchiectasis (44%) or COPD (56%) and in control subjects (10%). A diagnosis of gastroesophageal reflux was not associated with a higher concentration of EBC pepsin in bronchiectasis (P=.21) or COPD (P=.11). EBC pepsin concentration did not correlate with DMS (rs=0.36) or proximal reflux index (rs=0.25) in subjects with bronchiectasis or with DMS (rs=0.28) or proximal reflux index (rs=0.21) in patients with COPD. EBC and sputum pepsin concentrations were moderately correlated in bronchiectasis (rs=0.56) and in COPD (rs=0.43). CONCLUSIONS: Pepsin is detectable in EBC samples in bronchiectasis and COPD. Although no association was found between pepsin concentrations and a diagnosis of gastroesophageal reflux, a moderate relationship between sputum and EBC pepsin concentrations suggests that EBC pepsin may be a useful noninvasive marker of pulmonary microaspiration.

Proximal and distal gastro-oesophageal reflux in chronic obstructive pulmonary disease and bronchiectasis.

BACKGROUND AND OBJECTIVE: The aims of this observational study were (i) to examine the prevalence of symptomatic and clinically silent proximal and distal gastro-oesophageal reflux (GOR) in adults with chronic obstructive pulmonary disease (COPD) or bronchiectasis, (ii) the presence of gastric aspiration, and (iii) to explore the possible clinical significance of this comorbidity in these conditions. METHODS: Twenty-seven participants with COPD, 27 with bronchiectasis and 17 control subjects completed reflux symptom evaluation and dual-channel 24 h oesophageal pH monitoring. In those with lung disease, pepsin levels in sputum samples were measured using enzyme-linked immunosorbent assay, with disease severity (lung function and high-resolution computed tomography) also measured. RESULTS: The prevalence of GOR in COPD was 37%, in bronchiectasis was 40% and in control subjects was 18% (P = 0.005). Of those diagnosed with GOR, clinically silent reflux was detected in 20% of participants with COPD and 42% with bronchiectasis. While pepsin was found in 33% of COPD and 26% of bronchiectasis participants, the presence of pepsin in sputum was not related to a diagnosis of GOR based on oesophageal pH monitoring in either condition. Neither a diagnosis of GOR nor the presence of pepsin was associated with increased severity of lung disease in COPD or bronchiectasis. CONCLUSIONS: The prevalence of GOR in COPD or bronchiectasis is twice that of the control population, and the diagnosis could not be based on symptoms alone. Pepsin was detected in sputum in COPD and bronchiectasis, suggesting a possible role of pulmonary aspiration, which requires further exploration.

An acquired defect associated with abnormal signaling of the platelet collagen receptor glycoprotein VI.

INTRODUCTION: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. AIM: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. METHODS: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. RESULTS: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. CONCLUSIONS: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)ß(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbα, GPIbß, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbß is critical for ADAM17-dependent shedding of GPIbα induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbα and GPIbß subunits further lessened the restraint of GPIbß on GPIbα shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbß cytoplasmic domain disrupted calmodulin binding to GPIbß but had little effect on GPIbα shedding. Analysis of induction of GPIbα shedding by membrane-permeable GPIbß-derived peptides implicated the association of GPIbß with an unidentified intracellular protein in mediating regulation of GPIbα shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

Measuring soluble platelet glycoprotein VI in human plasma by ELISA.

Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcgammaRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of approximately 1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 +/- 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to approximately 7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was approximately 10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

Anti-glycoprotein VI monoclonal antibodies directly aggregate platelets independently of FcgammaRIIa and induce GPVI ectodomain shedding.

Adhesion of circulating platelets to the blood vessel wall initiates thrombus formation in haemostasis and thrombotic disease. The platelet collagen receptor, glycoprotein (GP) VI, is critical for thrombus formation at arterial shear rates and is a potential therapeutic target for anti-thrombotic drugs. In this study, we evaluate eight newly-derived, purified murine anti-human GPVI monoclonal antibodies (mAbs) for their effect on GPVI-dependent platelet aggregation and GPVI ectodomain shedding. All mAbs were raised against the ligand-binding GPVI ectodomain encompassing two immunoglobulin domains (residues 21-234, excluding the signal sequence) and recognized full-length GPVI in human platelet lysates by western blotting. The majority of antibodies induced aggregation in both human platelet-rich plasma (PRP) and washed platelets independently of the Fc receptor, FcgammaRIIa (not inhibited by the blocking anti-FcgammaRIIa mAb, IV.3), whereas one mAb (11A7) neither induced aggregation nor inhibited aggregation in response to GPVI ligands, collagen, and collagen-related peptide (CRP). In contrast, Fab fragments of mAb 12A5 strongly blocked collagen- and CRP-, but not convulxin-induced aggregation. In addition, it is shown for the first time in vitro that anti-GPVI mAbs can induce metalloproteinase-dependent ectodomain shedding of human GPVI, generating an approximately 10-kDa remnant that remained platelet-associated and an approximately 55-kDa soluble fragment. In conclusion, this analysis of anti-GPVI mAbs provides useful tools for studying the functional role of platelet GPVI.

The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2.

Angiotensin-converting enzyme (ACE)-2 is a homolog of the well-characterized plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome coronavirus. We have recently shown that like ACE, ACE2 undergoes ectodomain shedding and that this shedding event is up-regulated by phorbol esters. In the present study, we used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16-amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to glutathione-S-transferase-calmodulin, but not glutathione-S-transferase alone, in pull-down assays using cell lysates. Finally, to investigate whether calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin-specific inhibitors, trifluoperazine and calmidazolium. Both trifluoperazine (25 micromol/liter) and calmidazolium, (25 micromol/liter) significantly increased the release of ACE2 into the medium (44.1 +/- 10.8%, P < 0.05, Student's t test; unpaired, two-tailed, and 51.1 +/- 7.4% P < 0.05, one-way ANOVA, respectively;), as analyzed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin-specific inhibitor-stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.

A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex.

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3zeta-binding sites at the GPIb alpha C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)-dependent site on GPIb beta involving Ser166. 14-3-3zeta regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3zeta association blocks receptor signaling, suggesting a key functional role for 14-3-3zeta. We used deletion mutants of GPIb alpha expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3zeta binding to another GPIb-IX-V-associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)-PI3-kinase/p85-subunit and GST-14-3-3zeta indicated that both proteins interacted with contiguous GPIb alpha sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb alpha expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase-binding site. Pull-down experiments with GST-p85 truncates indicated the GPIb alpha-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3zeta-binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb alpha independently of 14-3-3zeta; 14-3-3zeta inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3zeta but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3zeta. Together, these data suggest the GPIb alpha C-terminus regulates signaling through independent association of 14-3-3zeta and PI3-kinase.

Dual ITAM-mediated proteolytic pathways for irreversible inactivation of platelet receptors: de-ITAM-izing FcgammaRIIa.

Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor gamma-chain (FcRgamma) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcgammaRIIa, also down-regulates GPVI expression. Agonists that signal through FcgammaRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcgammaRIIa also underwent rapid proteolysis in platelets treated with agonists for FcgammaRIIa (VM58/14A2) or GPVI/FcRgamma (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcgammaRIIa also bound calmodulin and W7 induced FcgammaRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcgammaRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, micro-calpain cleaved an FcgammaRIIa tail-fusion protein at (222)Lys/(223)Ala and (230)Gly/(231)Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRgamma and FcgammaRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcgammaRIIa after immunologic insult.

Calmodulin interacts with the platelet ADP receptor P2Y1.

P2Y1 [P2 (purinergic type-2)-receptor 1] is a G-protein-coupled ADP receptor that regulates platelet activation and ADP-induced Ca2+ signalling. Studies using P2Y1-knockout mice, G(q)-deficient mice or P2Y1-selective inhibitors have previously identified a key role for P2Y1 in pathophysiological thrombus formation at high shear stress. We provide evidence that a positively charged juxtamembrane sequence within the cytoplasmic C-terminal tail of P2Y1 can bind directly to the cytosolic regulatory protein calmodulin. Deletion by mutagenesis of the calmodulin-binding domain of P2Y1 inhibits intracellular Ca2+ flux in transfected cells. These results suggest that the interaction of calmodulin with the P2Y1 C-terminal tail may regulate P2Y1-dependent platelet aggregation.

Telomerase activation causes vascular smooth muscle cell proliferation in genetic hypertension.

Although abnormal cell growth in arterial vessel walls underpins vascular remodeling in high blood pressure, the molecular basis of the abnormality in hypertension has not been fully defined. Here, we report that in the aorta of spontaneously hypertensive rats, telomerase is selectively activated and telomeres are lengthened, in vivo and in vitro. Down-regulation of telomerase, the ribonucleoprotein complex responsible for the maintenance and elongation of telomeres (the ends of chromosomes) arrests the increased proliferation of spontaneously hypertensive rat vascular smooth muscle cells and induces apoptosis. This apoptosis is reversible by overexpressing telomerase and is prevented by increasing p53 tumor suppressor protein expression and worsened by lowering p53. Telomerase activation, telomere maintenance, and the p53 checkpoint appear to be critical for increased vascular smooth muscle proliferation, thus they represent potential novel therapeutic targets in hypertension.