Exhaled nitric oxide parameters and functional capacity in chronic obstructive pulmonary disease.

The extended exhaled nitric oxide (eNO) parameters, including peripheral or alveolar eNO, are investigational biomarkers in COPD. In this study, the hypothesis was tested that elevated peripheral eNO correlates with decreased functional capacity and lower global health status. Twenty-seven subjects with the Global Initiative for Chronic Obstructive Lung Disease stage 3 and 4 COPD were enrolled. Functional capacity and health status were tested using the 6 min walk test and St George's Respiratory Questionnaire (SGRQ) respectively. eNO parameters were estimated using multiple exhalation flow rates and were corrected for axial diffusion. The extended NO measurements were FE(NO)0.05 14.2 ppb (range 5.1-23.2), C(ANO) 4.6 ppb (2.2-6.9), D(awNO) 8.8 ml s(-1) (4.8-12.9), C(awNO) 83.2 ppb (29.9-128.7) and J'(awNO) 405 pl s(-1) (111-731). The distance traveled in the 6 min walk test was correlated with peripheral nitric oxide (r = -0.59, p = 0.03). SGRQ symptom score was correlated with maximum airway NO flux (r = -0.73, p = 0.01). SGRQ total score was correlated with maximum airway NO flux (r = -0.56, p = 0.05). In this study of subjects with severe COPD, peripheral nitric oxide correlated with functional capacity while large airway NO parameters correlated with symptom scores.

Phagocytosis of platelet microvesicles and beta2- glycoprotein I.

The majority of the antiphospholipid antibodies, present in patients with antiphospholipid syndrome, are directed against conformational epitopes in beta2-glycoprotein I. beta2-glycoprotein I is an anionic phospholipid-binding 50-kDa plasma protein whose physiological role is not clear. Here we investigate the role of beta2-glycoprotein I in the phagocytosis of phosphatidylserine-expressing platelet microvesicles and the effect of autoantibodies to beta2-glycoprotein I on this process. We labelled the glycans of beta2-glycoprotein I with BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-hydrazide without affecting its phospholipid binding capacity. BODIPY-beta2-glycoprotein I bound to platelet microvesicles in a concentration-dependent manner and promoted the phagocytosis of platelet microvesicles by THP-1 derived macrophages in vitro at physiological plasma concentrations with a half maximal effect at approximately 10 microg/ml. beta2-glycoprotein I-stimulated phagocytosis was inhibited by annexin A5 and the phosphatidylserine-binding C1C2 fragment of lactadherin. Furthermore, immunoaffinity purified beta2-glycoprotein I-dependent antiphospholipid antibodies from five patients with antiphospholipid syndrome inhibited the phagocytosis in a concentration-dependent manner. These studies suggest that the binding of beta2-glycoprotein I to phosphatidylserine-expressing procoagulant platelet microvesicles may promote their clearance by phagocytosis and autoantibodies to beta2-glycoprotein I may inhibit this process to induce a procoagulant state.

Platelet senescence and phosphatidylserine exposure.

BACKGROUND: The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (ΔΨm), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage. STUDY DESIGN AND METHODS: The exposure of phosphatidylserine and the loss of ΔΨm were determined in a flow cytometer using fluorescein isothiocyanate-lactadherin and JC-1, a lipophilic cationic reporter dye. The role of CypD was determined with cyclosporin A and CypD-deficient murine PLTs. Cytochrome c-induced caspase-3 and Rho-associated kinase I (ROCK1) activation were determined by immunoblotting and using their inhibitors. RESULTS: Collagen- and thrombin-induced exposure of phosphatidylserine was accompanied by a decrease in ΔΨm. Cyclosporin A inhibited the phosphatidylserine exposure and the loss of ΔΨm. CypD(-/-) mice had decreased loss of ΔΨm and impaired phosphatidylserine exposure. Collagen and thrombin did not induce the release of cytochrome c nor the activation of caspase-3 and ROCK1. In contrast, in PLTs stored for more than 5 days, the phosphatidylserine exposure was associated with cytochrome c-induced caspase-3 and ROCK1 activation. ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis, induced cytochrome c release and activation of caspase-3 and ROCK1 and phosphatidylserine exposure independent of CypD. CONCLUSION: These results show that in stored PLTs cytochrome c release and the subsequent activation of caspase-3 and ROCK1 mediate phosphatidylserine exposure and it is distinct from activation-induced phosphatidylserine exposure.

Lactadherin and clearance of platelet-derived microvesicles.

The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of the membrane bilayer during platelet activation is associated with the release of procoagulant phosphatidylserine-rich small membrane vesicles called platelet-derived microvesicles. We tested the effect of lactadherin, which promotes the phagocytosis of phosphatidylserine-expressing lymphocytes and red blood cells, in the clearance of platelet microvesicles. Platelet-derived microvesicles were labeled with BODIPY-maleimide and incubated with THP-1-derived macrophages. The extent of phagocytosis was quantified by flow cytometry. Lactadherin promoted phagocytosis in a concentration-dependent manner with a half-maximal effect at approximately 5 ng/mL. Lactadherin-deficient mice had increased number of platelet-derived microvesicles in their plasma compared with their wild-type littermates (950 +/- 165 vs 4760 +/- 650; P = .02) and generated 2-fold more thrombin. In addition, splenic macrophages from lactadherin-deficient mice showed decreased capacity to phagocytose platelet-derived microvesicles. In an in vivo model of light/dye-induced endothelial injury/thrombosis in the cremasteric venules, lactadherin-deficient mice had significantly shorter time for occlusion compared with their wild-type littermate controls (5.93 +/- 0.43 minutes vs 9.80 +/- 1.14 minutes;P = .01). These studies show that lactadherin mediates the clearance of phosphatidylserine-expressing platelet-derived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state.

Role of lactadherin in the clearance of phosphatidylserine-expressing red blood cells.

BACKGROUND: In red blood cells (RBCs) anionic phospholipids, such as phosphatidylserine, are present in the inner leaflet of the membrane bilayer. Exposure of phosphatidylserine occurs during senescence and during long-term storage of RBCs and is considered as the tag for removal from the circulation by macrophages. Lactadherin is a phosphatidylserine-binding glycoprotein secreted by macrophages that promotes the engulfment of phosphatidylserine-expressing apoptotic lymphocytes. This study investigates the role of lactadherin in the phagocytosis of phosphatidylserine-expressing RBCs. STUDY DESIGN AND METHODS: Transbilayer movement of phosphatidylserine was induced in RBCs either by storage beyond 30 days or by treatment with calcium ionophore A23187 and N-ethylmaleimide. Phosphatidylserine-expressing RBCs were incubated with phorbol ester-stimulated THP-1, and phagocytosis was determined by measuring the pseudoperoxidase activity of hemoglobin. The in vivo clearance of phosphatidylserine-enriched RBCs was measured in lactadherin-deficient mice and in their littermate controls. RESULTS: Lactadherin promoted phagocytosis of phosphatidylserine-expressing RBCs by macrophages in a concentration-dependent manner. Splenic macrophages from lactadherin-deficient mice had diminished capacity to phagocytose phosphatidylserine-expressing RBCs. The life span of RBCs in lactadherin-deficient mice was similar to wild-type littermate controls in vivo. However, when an excess of phosphatidylserine-expressing RBCs were infused, there was only a mild impairment in the clearance in lactadherin-deficient mice compared to wild-type littermate controls. CONCLUSION: These results show that clearance of phosphatidylserine-expressing RBCs is not diminished in a significant way in lactadherin-deficient mice under physiologic conditions and suggest the presence of redundant pathways.