The role of an enolase-related molecule in plasminogen binding to cells.

The alpha isoform of enolase is a candidate plasminogen receptor on U937 monocytoid cells [Miles, L. A., Dahlberg, C. L., Plescia, J., Felez, J., Kato, K. & Plow, E. F. (1991) Biochemistry 30, 1682-1691]. In the present study, an enolase-related molecule was detected on the surfaces of peripheral blood monocytes and neutrophils by fluorescence-activated cell sorting. A mRNA transcript encoding a unique membrane form of an enolase-related molecule was not detected by Northern-blotting and primer-extension analyses, consistent with the cell-surface protein being authentic alpha-enolase. Both the alpha and beta isoforms of purified enolase, bound plasminogen with an affinity similar to that of the cell surface. Moreover, immunopurified alpha-enolase enhanced plasminogen activation by tissue plasminogen activator and blocked the binding of plasminogen to alpha 2-antiplasmin, mimicking functions arising from the association of plasminogen with cells. The interaction of the enolase isoforms with plasminogen was dependent upon recognition of the C-terminal lysyl residue of the enolases by the lysine-binding sites of plasminogen, as the interaction was blocked by (a) peptides with C-terminal lysine residues and (b) an antibody to the C-terminal aspect of enolase. A monoclonal antibody was developed, characterized and utilized to quantify the enolase molecules present on the surface of U937 cells. A substantial number of molecules, 1.8 x 10(6)/cell, was present, accounting for approximately 10% of the plasminogen-binding capacity of these cells. These studies clearly establish the role of enolase as a cell-surface plasminogen-binding site with profibrinolytic functions.

Tissue factor mRNA in THP-1 monocytic cells is regulated at both transcriptional and posttranscriptional levels in response to lipopolysaccharide.

Tissue factor (TF) is transiently expressed in human monocytes exposed to the inflammatory agonist bacterial lipopolysaccharide (LPS). Since TF is the major cellular initiator of the coagulation protease cascades, it is inferred that its expression within the vasculature is strictly regulated. In this study, we investigated mechanisms which control TF mRNA expression in the human monocytic cell line THP-1. LPS induced a rapid and transient accumulation of the mature 2.2-kb TF mRNA, which was maximal at 2 h. After stimulation, the rate of transcription of the TF gene was increased (3.3 +/- 1.3)fold. In addition, we observed a significant change in TF mRNA stability: at 1 h after LPS stimulation, TF mRNA was stable during a 60-min period and had a half-life of greater than 120 min, whereas at 2 h, the half-life had declined to 25 +/- 5 min. Furthermore, a larger (3.4-kb) TF RNA species was induced in these cells; the size of this species and data from selective hybridizations with intron-specific probes are consistent with the presence of an unspliced copy of intron 1. These results demonstrate that the LPS-induced accumulation of TF mRNA levels in these monocytic cells is accomplished by both transcriptional and posttranscriptional control mechanisms.

Conservation of tissue factor primary sequence among three mammalian species.

Tissue factor (TF) is a transmembrane glycoprotein that serves as the cofactor for the initiation of the coagulation protease cascades. To identify conserved sequences of this molecule, a 1753-nucleotide cDNA encoding rabbit TF (rbTF) was isolated and sequenced. An open reading frame encoded a predicted precursor protein of 292 amino acids (aa), and a functionally active protein was synthesized when this cDNA was expressed in a eukaryotic cell system. The aa sequence of mature rbTF was 71% identical to human TF (huTF) and 58% to murine TF (muTF), consistent with the relative functional activity of each in human plasma. The structural organization of the protein was comparable in all three species, with a high degree of conservation of the extracellular domain, including the relative positions of cysteine residues and, to a lesser extent, the tripeptide motifs tryptophan-lysine-serine of huTF. In view of the uniform occurrence of TF functional activity throughout vertebrates, the sampling of these three distant mammalian species suggests that there is limited variance in primary sequence, consistent with the conserved function of TF.

Functional analysis of the human tissue factor promoter and induction by serum.

Tissue factor (TF) is the primary initiator of the coagulation protease cascades. This cell surface glycoprotein is the receptor and essential cofactor for the serine protease factor VIIa. TF is constitutively expressed in some extravascular cell types and is transiently induced in monocytes, endothelial cells, and fibroblasts. Inducible expression is implicated in cellular immune responses, inflammation, and intravascular coagulation. Transcriptional regulation of the TF promoter was analyzed in COS-7 cells under conditions of (i) high-level expression and (ii) serum induction. The region comprising nucleotides -209 to +121 (relative to the transcription start site) supports high-level transcriptional activity and can be divided into two distinct regions: a region (-111 to +121) that exhibited low promoter activity and a region (-209 to -112) that enhanced transcriptional activity to a high level. The role of further upstream sequences is still to be established, although two consensus binding sites for the transcriptional activator protein AP-1 did enhance low-level promoter activity. In serum-starved COS-7 cells TF expression was transiently increased 20-fold by serum. All transcriptionally active constructs were responsive to serum, indicating the presence of at least one serum response element, whose function was retained in the immediate 5' aspect of the gene, at -111 to +14. Based on this functional map, we propose that the elaborate pattern of TF expression by cells results from a relatively complex promoter.

Accidental industrial laser burn of macula.

A 20-year-old graduate student in physics was struck in the left eye by light from a dye laser. The injury resulted in rapid and profound loss of central visual acuity in the affected eye. Over the course of six months after the injury the corrected central vision improved to 20/25. Accidental retinal injuries due to laser light are discussed, as is the effect of nonionizing radiation on the retinal tissues.