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The cell surface receptor tissue factor (TF) binds to the serine protease coagulation factor VIIa. The binding sites are present from TF's concave side of the elbow angle at the module boundary to the convex side of the amino-terminal module at the edge of the three- and four-stranded beta-sheet. These ligand binding sites contribute one-third of the energy required for binding, while additional energy is contributed by the residues on the convex surface of the amino-terminal module.

A study of residue side chains reveals that the tissue factor extracellular domain possibly adopts the cytokine receptor homology unit folding pattern with two seven-beta-strand modules. Mutational analysis suggests that important residues are spatially closer and present as a cluster on the beta-strand module boundary. Residues involved in ligand binding occur in positions corresponding to ligand interactive residues.

A combinatorial library derived peptide, which binds to the extracellular domain of tissue factor, exhibits selective and quantitatively limited attenuation of factor X and the extrinsic limb of the coagulation protease cascades. Data indicate that such peptides provide attenuation of thrombotic risk on a long-term basis.

Macromolecular substrate docking that involves coagulation enzyme-cofactor complexes typically requires multiple contacts distant from an enzyme's catalytic cleft. To illustrate the role of the VIIa Gla-domain in macromolecular substrate docking, a group of site-directed mutants of this domain were generated and characterized. Analysis reveals that specific residues of the VIIa Gla-domain have a critical role in the activation of the macromolecular substrate factor X, one that is dependent on the Gla domain of the substrate being properly folded.

Abstract: RNA processing is vital for the high fidelity and diversity of eukaryotic transcriptomes and the encoded proteomes. However, control of RNA processing is not fully established. Σ RNA is a class of conserved large non-coding RNAs (murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense technology, we identified that RNA post-transcriptional modification is the most significant global function of Σ RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of Σ RNA/MALAT-1. These results support the hypothesis that Σ RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification. [Copyright &y& Elsevier]

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation. regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading. migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy. [ABSTRACT FROM AUTHOR]

The article discusses the findings of a study on the binding of coagulation protease factor XA to annexin 2 and the role of annexin 2 in regulating factor XA-mediated signal transduction independent of procoagulant activity. The authors state the enhanced interaction of factor Xa/annexin 2 in machrophages with lower tissue factor (TF) levels. The study explains the importance of autoproteolysis to binding. It reveals that the total level of factor Xa signaling remains unchanged in the presence of TF.

The conformation of the carboxy-terminal aspects of the Aα chain of human fibrinogen has been assessed by immunochemically characterizing the Aα 239 - 476 and Aα 518 - 584 regions of the molecule. Two peptides, corresponding to these regions, were isolated from cyanogen bromide digests of the Aα chain by molecular exclusion and high-performance liquid chromatography. Each pep tide reacted with antibodies elicited by immunization with the Aα chain and intact fibrinogen. Aα 239 - 476 appears to be a relatively immunodominant region of the molecule. Competitive inhibition analyses confirmed the accessibility of these regions to antibody in native fibrinogen. Each peptide, however, contained one or more epitopes, which was occult in the native molecule. These occult epitopes were expressed by the intact Aα chain and became accessible when fibrinogen was cleaved with plasmin. With plasmic degradation the epitopes expressed by fibrinogen and contained within these two peptide regions became significantly more reactive with antibody. This change occurred in concert with release of the Aα 518 - 584 region from the core of the molecule but did not require the generation of free Aα 239 - 476. Ultimately the epitopes within both regions were shed from the plasmic-resistant core of fibrinogen. Peptide epitopes were expressed in a similar manner by prolonged plasmic degradation of fibrinogen and fibrin with α chain cross-linking. These results are generally consistent with models depicting the carboxy-terminal aspects of the Aα chain as being surface-oriented but suggest a systematic ordering of structure when these regions are integrated into the native molecule. Plasmic cleavage significantly relaxes the conformational restraints on the organization within this region. [ABSTRACT FROM AUTHOR]

Circulating monocytes as well as resident monocyte-derived macrophages (MDMs) in the atherosclerotic plaque express tissue factor. We tested the hypothesis that differentiation of monocytes into macrophages increases their tissue factor expression. This was done by isolating monocytes from human blood and culturing them for 24 h or allowing their differentiation into macrophages for 8 days before assay. Tissue factor procoagulant activity was assessed by recalcification clotting assay while Western blotting quantitated tissue factor protein. Immunocytochemistry was applied to localize antigenic tissue factor in cells. The results showed that freshly-isolated monocytes expressed low baseline procoagulant activity (0.0004±0.0003 ng rhTF/106 cells) which increased 100-fold in 24-h cultured monocytes (0.04±0.01 ng/106 cells, P<0.0001) and was associated with detectable tissue factor protein by Western blotting. Furthermore, MDMs expressed 25-fold higher procoagulant activity (1.0±0.5 ng/106 cells, P<0.04) and increased tissue factor protein content compared with 24-h monocytes as corroborated by immunocytochemistry. LPS stimulation increased baseline binding activity of transcription factor κB three-fold (P<0.03) and 2.6-fold (P<0.09) in monocytes and in macrophages, respectively. The results demonstrate that differentiation of human monocytes into macrophages in culture enhances their tissue factor expression. The observed tissue factor increase that can be further stimulated by LPS may contribute to thrombogenicity of macrophage-rich atheroma. Activation of the TF-κB transduction pathway suggests its potential role in the transcriptional regulation of TF gene in response to acute stimulation in these cells. This cell-culture model may provide the opportunity to evaluate the effect of various interventions on macrophage procoagulant activity. [Copyright &y& Elsevier]