Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The fie gene was located in chromosomal region ip33 to ip34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts oftie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.

The P-selectin counterreceptor PSGL-1 is covalently modified by mono ␣2,3-sialylated, multiply ␣1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte ␣1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors. We find that Fuc-TIV can transfer fucose effectively to all N-acetyllactosamine (LN) units in neutral polylactosamines, and to the "inner" LN units of ␣2,3-sialylated acceptors but is ineffective in transfer to the distal ␣2,3-sialylated LN unit in ␣2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal ␣2,3-sialylated LN unit in ␣2,3-sialylated acceptors and thus exhibits an acceptor site-specificity that is complementary to Fuc-TIV. Furthermore, the consecutive action of Fuc-TIV and Fuc-TVII, in vitro, can convert the long chain sialoglycan SA␣2-3LN␤1-3LN␤1-3LN (where SA is sialic acid) into the trifucosylated molecule SA␣2-3Lex␤1-3Lex␤1-3Lex (where Lex is the trisaccharide Gal␤1-4(Fuc␣1-3)GlcNAc) known to decorate PSGL-1. The complementary in vitro acceptor site-specificities of Fuc-TIV and Fuc-TVII imply that these enzymes cooperate in vivo in the biosynthesis of monosialylated, multifucosylated polylactosamine components of selectin counterreceptors on human leukocytes.

Increased mortality among adult asthmatics was largely explained by the development of COPD, malignant respiratory tract neoplasms, and cardiovascular diseases. Smoking cessation is important for reduction in total mortality in both asthmatic and nonasthmatic adults. AR/AC was associated with decreased mortality only in asthmatics. Thus, studies in other populations of larger size are needed to explore further the nature of this association.