Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity.

Summary.A woman with a preliminary diagnosis of afibrinogenaemia was later found to have a functional fibrinogen of 0 . 06 mg/ml and markedly prolonged thrombin and reptilase times. The stoichiometry of fibrinopeptide release was normal but there was a gross delay in the polymerization of purified fibrin. Plasma protein electrophoresis showed an absence of normal fibrinogen and a novel anodal component which was confirmed as fibrinogen by immunofixation. Western blots of non-reducing SDS-PAGE gels indicated a molecular weight of 270 kD, compared to 340 kD for normal fibrinogen and similar analysis of reducing gels showed that the expected 67 kD Aa chain was missing and replaced by a 30 kD band. This aberrant chain was not detected by the monoclonal antibody F-103, which recognizes the epitope formed by residues 259-276 of the Aa chain. Cycle sequencing of the DNA encoding the F-103 epitope revealed the homozygous insertion of cytosine at position 4133 of the gene sequence. Predictably this translates as three new amino acids ( 268 Gln-Glu-Pro) before termination at a new (TAG) stop codon. No abnormal Aa chains could be detected in plasma from the woman's heterozygous son. The hypofibrinogenaemia observed is likely to be the result of diminished assembly and/or secretion of the truncated Aa chains rather than enhanced extracellular degradation.

A study of blood lead levels and intelligence, reading, and behaviour problems was carried out using a sample of 579 Dunedin 11-yr-old children. The results suggested that when account was taken of social, environmental, and background factors, raised blood lead is associated with a small but statistically significant increase in children's general behaviour problems as reported by both parents and teachers. These results applied especially to the more specific problems of inattention and hyperactivity.

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We investigated the molecular basis of hypofibrinogenemia in a man with a normal thrombin clotting time. Protein analysis indicated equal plasma expression of 2 different Bβ alleles, and DNA sequencing confirmed heterozygosity for a new Bβ235 P→L mutation. Protein analysis also revealed a novel γD chain, present at a ratio of 1:2 relative to the γA chain. Mass spectrometry indicated a 14 d decrease in the γD-chain mass, and DNA sequencing showed this was caused by a novel γ82 A→G substitution. DNA sequencing established heterozygosity for 2 further mutations: T→C in intron 4 of the A gene and A→C in the 3′ noncoding region of the Bβ gene. Studies on the man's daughter, together with plasma expression levels, discounted both the A and Bβ mutations as the cause of the low fibrinogen, suggesting that the γ82 mutation caused the hypofibrinogenemia. This was supported by analysis of 31 normal controls in whom the Bβ mutations were found at polymorphic levels, with an allelic frequency of 5% for the Bβ235 mutation and 42% for the Bβ 3′ untranslated mutation. The γ82 mutation was, however, unique to the propositus. Residue γ82 is located in the triple helix that separates the E and D domains, and aberrant packing of the helices may explain the decreased fibrinogen concentration.